USE OF RNASE H FOR THE SELECTIVE AMPLIFICATION OF VIRAL DNA

FIELD OF THE INVENTION

The present invention belongs to the field of in- vitro diagnostics. Within this field, it particularly concerns the specific amplification of a DNA target nucleic acid that may be present in a sample with reduced or abolished co-amplification of RNA nucleic acids present in said sample.

BACKGROUND OF THE INVENTION

In the field of molecular diagnostics, the amplification of nucleic acids from numerous sources has been of considerable significance. Examples for diagnostic applications of nucleic acid amplification and detection are the detection of viruses such as Human Papilloma Virus (HPV), West Nile Virus (WNV) or the routine screening of blood donations for the presence of Human Immunodeficiency Virus (HIV), Hepatitis-B (HBV) and/or C Virus (HCV). Furthermore, said amplification techniques are suitable for bacterial targets such as mycobacteria, or the analysis of oncology markers.

The most prominent and widely-used amplification technique is Polymerase Chain Reaction (PCR). Other amplification reactions comprise, among others, the Ligase Chain Reaction, Polymerase Ligase Chain Reaction, Gap-LCR, Repair Chain Reaction, 3SR, NASBA, Strand Displacement Amplification (SDA), Transcription Mediated Amplification (TMA), and Q - amplification.

Automated systems for PCR-based analysis often make use of real-time detection of product amplification during the PCR process in the same reaction vessel. Key to such methods is the use of modified oligonucleotides carrying reporter groups or labels.

It has been shown that amplification and detection of more than one target nucleic acid in the same vessel is possible. This method is commonly termed "multiplex" amplification and requires different labels for distinction if real-time detection is performed.

It is mostly desirable or even mandatory in the field of clinical nucleic acid diagnostics to control the respective amplification using control nucleic acids with a known sequence, for qualitative (performance control) and/or quantitative (determination of the quantity of a target nucleic using the control as a reference) purposes. Given the diversity especially of diagnostic targets, comprising prokaryotic, eukaryotic as well as viral nucleic acids, and given the diversity between different types of nucleic acids such as RNA and DNA, control nucleic acids are usually designed in a specific manner. In brief, these controls usually resemble the target nucleic acid for which they serve as control in order to mimic their properties during the process. This circumstance applies for both qualitative and quantitative assays. In case multiple parameters are to be detected in a single or in parallel experiments, usually different controls resembling different target nucleic acids are employed, such as e.g. in Swanson et al. (J. Clin. Microbiol, (2004), 42, pp. 1863-1868). Stocher et al. (J. Virol. Meth. (2003), 108, pp. 1-8) discloses a control nucleic acid in which multiple virus-specific competitive controls are comprised on the same DNA molecule.

The present invention provides a controlled amplification method using a different approach that displays various advantages. SUMMARY OF THE INVENTION

In one aspect the invention relates to a process for amplifying a DNA target nucleic acid that may be present in a sample, the process comprising the steps of contacting the DNA target nucleic acid from the sample with an amplification reagent comprising at least one pair of oligonucleotide primers that specifically hybridizes under suitable conditions to said DNA target nucleic acid, an RNase H and a polymerase with reverse transcriptase activity; incubating in a reaction vessel the DNA target nucleic acid with the amplification reagent for a period of time and under conditions suitable for transcription of RNA by the polymerase with reverse transcriptase activity to occur; and incubating in the reaction vessel the DNA target nucleic acid with the amplification reagent for a period of time and under conditions suitable for an amplification reaction indicative of the presence or the absence of the DNA target nucleic acid to occur. In some embodiments said polymerase with reverse transcriptase activity is a polymerase comprising a mutation conferring an improved reverse transcriptase activity relative to a respective wildtype polymerase. In some embodiments said polymerase with reverse transcriptase activity comprising a mutation is derived from a polymerase selected from the group consisting of a CS5 DNA polymerase, a CS6 DNA polymerase, a Thermotoga maritima DNA polymerase, a Thermus aquaticus DNA polymerase, a Thermus thermophilus DNA polymerase, a Thermus flavus DNA polymerase, a Thermus filiformis DNA polymerase, a Thermus sp. spsl7 DNA polymerase, a Thermus sp. Z05 DNA polymerase, a Thermotoga neapolitana DNA polymerase, a Termosipho africanus DNA polymerase, and a Thermus caldophilus DNA polymerase. In some embodiments said polymerase with reverse transcriptase activity comprising a mutation is derived from a Thermus sp. Z05 DNA polymerase. In some embodiments the process further comprises amplifying an RNA target nucleic acid that may be present in said sample. In some embodiments said DNA target nucleic acid is derived from Hepatitis B Virus (HBV). In some embodiments said sample is a fluid sample. In some embodiments the amplification reagent further comprises any one of: a buffer solution comprising salts suitable for performing an amplification reaction, mononucleotides such as nucleoside triphosphates, oligonucleotide primers, oligonucleotide detection probes, and/or dyes. In one embodiment, the process is an automated process.

In another aspect the invention relates to a process for amplifying and determining the quantity of a DNA target nucleic acid that may be present in a sample, said process comprising the steps of incubating in a reaction vessel said DNA target nucleic acid from the sample with an amplification reagent comprising at least one pair of oligonucleotide primers that specifically hybridizes under suitable conditions to said DNA target nucleic acid, RNase H and a polymerase with reverse transcriptase activity, for a period of time and under conditions suitable for transcription of RNA by said polymerase with reverse transcriptase activity to occur; incubating in the reaction vessel the DNA target nucleic acid with said amplification reagent comprising said oligonucleotide primers specific for the DNA target nucleic acid for a period of time and under conditions suitable for an amplification reaction indicative of the presence or absence of the DNA target nucleic acid to occur; and determining the quantity of the DNA target nucleic acid by referencing to an external calibration or by implementing an internal quantitative standard. In some embodiments said polymerase with reverse transcriptase activity is a polymerase comprising a mutation conferring an improved reverse transcriptase activity relative to a respective wildtype polymerase. In some embodiments said polymerase with reverse transcriptase activity comprising a mutation is selected from the group consisting of a CS5 DNA polymerase, a CS6 DNA polymerase, a Thermotoga maritima DNA polymerase, a Thermus aquaticus DNA polymerase, a Thermus thermophilus DNA polymerase, a Thermus flavus DNA polymerase, a Thermus filiformis DNA polymerase, a Thermus sp. spsl7 DNA polymerase, a Thermus sp. Z05 DNA polymerase, a Thermotoga neapolitana DNA polymerase, a Termosipho africanus DNA polymerase, and a Thermus caldophilus DNA polymerase. In some embodiments said polymerase with reverse transcriptase activity comprising a mutation is from Thermus sp. Z05 DNA polymerase. In some embodiments the process further comprises amplifying a RNA target nucleic acid that may be present in said sample. In some embodiments said DNA target nucleic acid is Hepatitis B Virus (HBV). In some embodiments said sample is a fluid sample. In some embodiments the amplification reagent further comprises any one of: a buffer solution comprising salts suitable for performing an amplification reaction, mononucleotides such as nucleoside triphosphates, at least one additional oligonucleotide primer, at least one oligonucleotide detection probe, and/or dyes. In one embodiment, the process is an automated process.

DESCRIPTION OF THE INVENTION

In certain amplification reactions of DNA nucleic acids potentially present in a biological sample co-amplification of RNA nucleic acids may occur thereby posing a risk of false positive signal determination for the DNA nucleic acid and/or false quantification of the DNA nucleic acid. Hence, there is a need in in vitro diagnostic applications to reduce or abolish co- amplification of RNA nucleic acids present in said sample to improve accuracy of quantitative and/or qualitative determinations of DNA target nucleic acids in biological samples.

The problem solved by the invention therefore lies in the reduction or abolishment of "unwanted" co-amplification of RNA nucleic acids derived from a DNA target nucleic acid (e.g. amplification of viral RNA also present in a sample that contains viral DNA when only viral DNA amplification is desired) by utilizing a polymerase having reverse transcriptase activity and adding RNase H to the reaction mixture for performing the amplification reaction. Herein, the reaction mixture is subjected to conditions suitable for transcription of RNA templates to occur by the polymerase having reverse transcriptase activity resulting in the reverse transcription of the RNA template and the formation of RNA-DNA hybrids. Concurrently, the RNase H present in the reaction mixture degrades such RNA-DNA hybrids so that these hybrids no longer exist as templates for amplification and only the "pure" DNA target will be amplified. Thus, the risk of false positive signal determination for the DNA target nucleic acid and/or of false quantification of the DNA target nucleic acid are vastly reduced or completely abolished.

In one aspect a process for amplifying a DNA target nucleic acid that may be present in a sample is provided comprising the steps of contacting the DNA target nucleic acid from the sample with an amplification reagent comprising at least one pair of distinct oligonucleotide primers that specifically hybridizes under suitable conditions to said DNA target nucleic acid, an RNase H and a polymerase with reverse transcriptase activity; incubating in a reaction vessel the DNA target nucleic acid with the amplification reagent for a period of time and under conditions suitable for transcription of RNA by the polymerase with reverse transcriptase activity to occur; and incubating in the reaction vessel the DNA target nucleic acid with the amplification reagent for a period of time and under conditions suitable for an amplification reaction indicative of the presence or the absence of the DNA target nucleic acid to occur.

In another aspect a process for amplifying and determining the quantity of a DNA target nucleic acid that may be present in a sample is provided that comprises the steps of incubating in a reaction vessel said DNA target nucleic acid from the sample with an amplification reagent comprising at least one pair of distinct oligonucleotide primers that specifically hybridizes under suitable conditions to said DNA target nucleic acid, RNase H and a polymerase with reverse transcriptase activity, for a period of time and under conditions suitable for transcription of RNA by said polymerase with reverse transcriptase activity to occur; incubating in the reaction vessel the DNA target nucleic acid with said amplification reagent comprising said distinct oligonucleotide primers specific for the DNA target nucleic acid for a period of time and under conditions suitable for an amplification reaction indicative of the presence or absence of the DNA target nucleic acid to occur; and determining the quantity of the DNA target nucleic acid by referencing to an external calibration or by implementing an internal quantitative standard.

In another aspect a process for isolating and amplifying at least one target nucleic acid that may be present in a sample is provided comprising the steps of: combining together a solid support material and the sample in a reaction vessel for a period of time and under conditions suitable to permit nucleic acids comprising the target nucleic acids to be immobilized on the solid support material; isolating the solid support material from the other material present in the sample; purifying the nucleic acids bound to the solid support material and washing the solid support material one or more times with a wash buffer; contacting in a reaction vessel the purified target nucleic acids with an amplification reagent comprising at least one set of distinct oligonucleotide primers for the target nucleic acid, RNase H and a polymerase with reverse transcriptase activity to form a reaction mixture; incubating said reaction mixture in the reaction vessel for a period of time and under conditions suitable for transcription of RNA by said polymerase with reverse transcriptase activity to occur; incubating said reaction mixture in the reaction vessel for a period of time and under conditions suitable for an amplification reaction indicative of the presence or absence of the DNA target nucleic acid to occur; detecting and measuring a signal generated by the amplification products of said target nucleic acid indicative for the presence or absence of the target nucleic acid in the sample. In certain aspects the process may be an automated process. Herein, in certain aspects the isolation step is performed in a separation station. In another aspect a process for isolating and amplifying at least one target nucleic acid that may be present in a sample is provided comprising the steps of: adding an internal control nucleic acid to said sample; combining together a solid support material and the sample in a reaction vessel for a period of time and under conditions suitable to permit nucleic acids comprising the target nucleic acids and the internal control nucleic acid to be immobilized on the solid support material; isolating the solid support material from the other material present in the sample; purifying the nucleic acids bound to the solid support material and washing the solid support material one or more times with a wash buffer; contacting in a reaction vessel the purified target nucleic acids and the purified internal control nucleic acid with an amplification reagent comprising at least one set of distinct oligonucleotide primers for the target nucleic acid and one set of oligonucleotide primers for said internal control nucleic acid in the reaction vessels, RNase H and a polymerase with reverse transcriptase activity to form a reaction mixture; incubating said reaction mixture in the reaction vessel for a period of time and under conditions suitable for transcription of RNA by said polymerase with reverse transcriptase activity to occur; incubating said reaction mixture in the reaction vessel for a period of time and under conditions suitable for an amplification reaction indicative of the presence or absence of the DNA target nucleic acid to occur; detecting and measuring a signal generated by the amplification products of said target nucleic acid and being proportional to the concentration of said target nucleic acid, and detecting and measuring a signal generated by said internal control nucleic acid. In certain aspects the process may further comprise determining the quantity of the target nucleic acid by referencing the signal measured for the amplified products of said target nucleic acid to the signal measured for the internal control nucleic acid. In certain aspects the process may be an automated process. Herein, in certain aspects the isolation step is performed in a separation station.

In another aspect a process for isolating and simultaneously amplifying at least a first and a second target nucleic acid that may be present in one or more samples is provided, said process comprising the automated steps of:

a. adding an internal control nucleic acid to each of said samples;

b. combining together a solid support material and said one or more samples in one or more vessels for a period of time and under conditions suitable to permit nucleic acids comprising the target nucleic acids and the internal control nucleic acid to be immobilized on the solid support material;

c. isolating the solid support material from the other material present in the samples in a separation station; d. purifying the nucleic acids in said separation station and washing the solid support material one or more times with a wash buffer;

e. contacting the purified target nucleic acids and the purified internal control nucleic acid with an amplification reagent comprising R ase H, a polymerase with reverse transcriptase activity and at least one distinct set of oligonucleotide primers for each of said target nucleic acids and for said internal control nucleic acid in at least two reaction vessels, wherein at least a first reaction vessel comprises at least said first target nucleic acid and at least a second reaction vessel comprises at least said second target nucleic acid and wherein the second target nucleic acid is absent from the first reaction vessel;

f. incubating in the reaction vessels said purified target nucleic acids and said purified internal control nucleic acid with said amplification reagent for a period of time and under conditions suitable for transcription of RNA by said polymerase with reverse transcriptase activity to occur;

g. incubating in said reaction vessel said purified target nucleic acids and said purified internal control nucleic acid with said amplification reagent for a period of time and under conditions suitable for an amplification reaction indicative of the presence or absence of said target nucleic acids to occur;

h. detecting and measuring signals generated by the amplification products of said target nucleic acids and being proportional to the concentration of said target nucleic acids, and detecting and measuring a signal generated by said internal control nucleic acid, wherein the conditions for amplification and detection in steps d. to h. are identical for said at least first and second purified target nucleic acids and said internal control nucleic acid, and wherein the sequence of said internal control nucleic acid is identical for said at least first and second purified target nucleic acids.

In some aspects of the above processes said polymerase with reverse transcriptase activity is a polymerase comprising a mutation conferring an improved reverse transcriptase activity relative to a respective wildtype polymerase. In some aspects said polymerase with reverse transcriptase activity comprising a mutation is derived from a polymerase selected from the group consisting of a CS5 DNA polymerase, a CS6 DNA polymerase, a Thermotoga maritima DNA polymerase, a Thermus aquaticus DNA polymerase, a Thermus thermophilus DNA polymerase, a Thermus flavus DNA polymerase, a Thermus filiformis DNA polymerase, a Thermus sp. spsl7 DNA polymerase, a Thermus sp. Z05 DNA polymerase, a Thermotoga neapolitana DNA polymerase, a Termosipho africanus DNA polymerase, and a Thermus caldophilus DNA polymerase. In some aspects said polymerase with reverse transcriptase activity comprising a mutation is derived from a Thermus sp. Z05 DNA polymerase. In some aspects the process further comprises amplifying an RNA target nucleic acid that may be present in said sample. In some aspects said DNA target nucleic acid is derived from Hepatitis B Virus (HBV). In some aspects said sample is a fluid sample. In some aspects the amplification reagent further comprises any one of: a buffer solution comprising salts suitable for performing an amplification reaction, mononucleotides such as nucleoside triphosphates, oligonucleotide primers, oligonucleotide detection probes, and/or dyes. In some aspects the process is an automated process.

The present disclosure further allows for the development of simultaneous assays on a plurality of parameters and/or nucleic acid types while using the same internal control nucleic acid sequence for said different parameters and/or nucleic acid types. Therefore, it contributes to reducing the overall complexity of the corresponding experiments on various levels: For instance, only one internal control nucleic acid sequence has to be designed and added to the respective amplification mixes, thus saving the time and costs for designing and synthesizing or buying multiple control nucleic acid sequences. The assay or assays can be streamlined, and the risk of handling errors is reduced. In addition, the more different control nucleic acid sequences are employed in one assay or parallel assays carried out simultaneously under the same conditions, the more complex it may result to adjust the respective conditions. Moreover, with a single control suitable for a plurality of nucleic acids, said control can be dispensed from a single source e.g. into different vessels containing said different target nucleic acids. Within the scope of the disclosure, the single control nucleic acid sequence may also serve as a qualitative and as a quantitative control.

As a further advantage of the method described above, the testing of a particular biological sample for other nucleic acids in possible subsequent experiments need not involve another sample preparation procedure with the addition of a different internal control nucleic acid, since the control used can be used to control the amplification of different nucleic acids. Thus, once an internal control nucleic acid has been added, other parameters may be tested in the same sample under the same conditions.

The internal control nucleic acid can be competitive, non-competitive or partially competitive. A competitive internal control nucleic acid carries essentially the same primer binding sites as the target and thus competes for the same primers with the target. While this principle allows a good mimicry of the respective target nucleic acid due to their similar structure, it can lower the amplification efficiency with regard to the target nucleic acid or acids and thus lead to a less sensitive assay.

A non-competitive internal control nucleic acid has different primer binding sites than the target and thus binds to different primers. Advantages of such a setup comprise, among others, the fact that the single amplification events of the different nucleic acids in the reaction mixture can take place independently from each other without any competition effects. Thus, no adverse effects occur regarding the limit of detection of the assay as can be the case in a competitive setup.

Finally, in an amplification using a partially competitive setup the respective control nucleic acid and at least one of the target nucleic acids compete for the same primers, while at least one other target nucleic acid binds to different primers.

The fact that the methods described above involve a distinct set of primers for each of said target nucleic acids and for said internal control nucleic acid renders the method considerably flexible. In this non-competitive setup it is not necessary to introduce target-specific binding sites into the control nucleic acid as in the case of a competitive setup, and the drawbacks of a competitive setup as mentioned above are avoided. In a non-competitive setup, the internal control nucleic acid has a sequence different from any target sequences, in order not to compete for their primers and/or probes. For example, the sequence of the internal control nucleic acid can be different from the other nucleic acid sequences in the fluid sample. As an example, if the fluid sample is derived from a human, the internal control nucleic acid may not have a sequence which also endogenous ly occurs within humans. The difference in sequence should thus be at least significant enough to not allow the binding of primers and/or probes to the respective endogenous nucleic acid or acids under stringent conditions and thus render the setup competitive. In order to avoid such interference, the sequence of the internal control nucleic acid used can be derived from a source different from the origin of the fluid sample. For example, it is derived from a naturally occurring genome, for example a plant genome, or from a grape genome. In an embodiment, a nucleic acid derived from a naturally occurring genome is scrambled. As known in the art, "scrambling" means introducing base mutations in a sequence to a certain extent. For example, the sequence of the internal control nucleic acid used is substantially altered with respect to the naturally occurring gene it is derived from.

The process comprising the automated steps mentioned above also displays various additional advantages: It has been a challenge in the prior art that the number of different target nucleic acids in a multiplex assay carried out in a single reaction vessel is limited by the number of appropriate labels. In a real-time PCR assay, for example, the potential overlap of fluorochrome spectra has a great impact on assay performance (risk of false positive results, lower precision etc.) Therefore, the respective fluorophores have to be carefully selected and spectrally well separated in order to assure the desired performance of a diagnostic test. Typically, the number of different usable fluorophores corresponds to a single-digit number of PCR instrument fluorescence channels.

In contrast, in the process described supra, the internally controlled amplification of at least a first and a second target nucleic acid takes place in at least two different reaction vessels, allowing for the simultaneous amplification of a higher number of different target nucleic acids, since signals in different reaction vessels can be detected independently from each other. Still, embodiments are disclosed wherein in one or more of the multiple reaction vessels multiplex reactions are performed, thereby multiplying the number of targets that may be amplified simultaneously and under the same conditions. In such embodiments, the internal control nucleic acid serves as a control for the different target nucleic acids within a vessel as well as different target nucleic acids in different vessel.

Thus, one aspect of the disclosure relates to the process described supra, wherein at least two target nucleic acids are amplified in the same reaction vessel.

For example, it may be preferred to amplify the first, but not the second target nucleic acid in the first reaction vessel and only the second, but not the first target nucleic acid in the second reaction vessel, e.g. depending on the sample and/or the target nucleic acid or acids in question.

Therefore, further disclosed is the process described above, wherein the first target nucleic acid is absent from the second reaction vessel.

Especially if a fluid sample is suspected to contain target nucleic acids from different organisms, or even the different organisms as such, or if it is not clear which of the different nucleic acids or organisms may be present in said sample, an advantageous embodiment of the disclosure is the process described above, wherein the first target nucleic acid and the second target nucleic acid are from different organisms.

A further aspect of the disclosure is the process described above, wherein the first and/or the second target nucleic acid is a non- viral nucleic acid.

Also, an aspect of the disclosure is the process described supra, wherein the first and/or the second target nucleic acid is a bacterial nucleic acid. As described before, the method described above is useful for qualitatively or quantitatively controlling the amplification of at least a first and a second target nucleic acid.

Qualitative detection of a nucleic acid in a biological sample is crucial e.g. for recognizing an infection of an individual. Thereby, one important requirement for an assay for detection of a microbial infection is that false-negative or false-positive results be avoided, since such results would almost inevitably lead to severe consequences with regard to treatment of the respective patient. Thus, especially in PCR-based methods, a qualitative internal control nucleic acid is added to the detection mix. Said control is particularly important for confirming the validity of a test result: At least in the case of a negative result with regard to the respective target nucleic acid, the qualitative internal control reaction has to perform reactive within given settings, i.e. the qualitative internal control must be detected, otherwise the test itself is considered to be inoperative. However, in a qualitative setup, said qualitative internal control does not necessarily have to be detected in case of a positive result. For qualitative tests, it is especially important that the sensitivity of the reaction is guaranteed and therefore strictly controlled As a consequence, the concentration of the qualitative internal control must be relatively low so that even in a situation e.g. of slight inhibition the qualitative internal control is not be detected and therefore the test is invalidated.

Thus, an aspect of the disclosure is the process described above, wherein the presence of an amplification product of said internal control nucleic acid is indicative of an amplification occurring in the reaction mixture even in the absence of amplification products for one or more of said target nucleic acids.

On the other hand and in addition to mere detection of the presence or absence of a nucleic acid in a sample, it is often important to determine the quantity of said nucleic acid. As an example, stage and severity of a viral disease may be assessed on the basis of the viral load. Further, monitoring of any therapy requires information on the quantity of a pathogen present in an individual in order to evaluate the therapy's success. For a quantitative assay, it is necessary to introduce a quantitative standard nucleic acid serving as a reference for determining the absolute quantity of a target nucleic acid. Quantitation can be effectuated either by referencing to an external calibration or by implementing an internal quantitative standard.

In the case of an external calibration, standard curves are created in separate reactions using known amounts of identical or comparable nucleic acids. The absolute quantity of a target nucleic acid is subsequently determined by comparison of the result obtained with the analyzed sample with said standard function. External calibration, however, has the disadvantage that a possible extraction procedure, its varied efficacy, and the possible and often not predictable presence of agents inhibiting the amplification and/or detection reaction are not reflected in the control.

This circumstance applies to any sample-related effects. Therefore, it might be the case that a sample is judged as negative due to an unsuccessful extraction procedure or other sample- based factors, whereas the target nucleic acid to be detected and quantified is actually present in the sample.

For these and other reasons, an internal control nucleic acid added to the test reaction itself is of advantage. When serving as a quantitative standard, said internal control nucleic acid has at least the following two functions in a quantitative test:

i) It monitors the validity of the reaction.

ii) It serves as reference in titer calculation thus compensating for effects of inhibition and controlling the preparation and amplification processes to allow a more accurate quantitation. Therefore, in contrast to the qualitative internal control nucleic acid in a qualitative test which must be positive only in a target-negative reaction, the quantitative control nucleic acid in a quantitative test has two functions: reaction control and reaction calibration. Therefore it must be positive and valid both in target-negative and target-positive reactions.

It further has to be suited to provide a reliable reference value for the calculation of high nucleic acid concentrations. Thus, the concentration of an internal quantitative control nucleic acid needs to be relatively high.

Therefore, in another aspect the process described above further comprises the following step: i. determining the quantity of one or more of said target nucleic acids.

The internally controlled process described above requires considerably less hands-on time and testing is much simpler to perform than real-time PCR methods used in the prior art. The process offers a major advantage e.g. in the field of clinical virology as it permits parallel amplification of several viruses in parallel experiments. The process is particularly useful in the management of post-transplant patients, in whom frequent viral monitoring is required. Thereby said process facilitates cost-effective diagnosis and contributes to a decrease in the use of antiviral agents and in viral complications and hospitalizations. This equally applies to the field of clinical microbiology. In general, efficiencies will be gained in faster turnaround time and improved testing flexibility. Consequently, this leads to a decrease in the number of tests requested on a patient to make a diagnosis, and potentially shorter hospital stays (e.g. if a diagnosis can be provided sooner, patients requiring antimicrobial therapy will receive it sooner and thus recover earlier). In addition, patients show less morbidity and therefore cause fewer costs related to supportive therapy (e.g., intensive care related to a delay in diagnosis of sepsis). Providing a negative result sooner can have important implications for the overprescription of antibiotics. For example, if a test result obtained by the process according to the disclosure is able to rule out the pathogen more quickly than with a standard real-time PCR method, then the clinician will not be forced to use empirical antibiotics. Alternatively, if empirical antibiotics are used, the duration of the respective treatment can be shortened.

With respect to designing a specific test based on the process according to the disclosure, the skilled artisan will particularly, but not only, benefit from the following advantages:

• a reduction in software complexity (leading to a reduced risk of programming errors) · focusing of assay development efforts on the chemistry optimization instead of the chemistry plus the instrument control parameters

• much more reliable system since a single process is always used and the hardware can be optimally designed to perform this protocol

• the skilled artisan performing the internally controlled process described above is provided with the flexibility to run multiple different assays in parallel as part of the same process

• cost reduction.

In the sense of the disclosure, "purification", "isolation" or "extraction" of nucleic acids relate to the following: Before nucleic acids may be analyzed in a diagnostic assay e.g. by amplification, they typically have to be purified, isolated or extracted from biological samples containing complex mixtures of different components. Often, for the first steps, processes are used which allow the enrichment of the nucleic acids. To release the contents of cells or viral particles, they may be treated with enzymes or with chemicals to dissolve, degrade or denature the cellular walls or viral particles. This process is commonly referred to as lysis. The resulting solution containing such lysed material is referred to as lysate. A problem often encountered during lysis is that other enzymes degrading the component of interest, e.g. deoxyribonucleases or ribonucleases degrading nucleic acids, come into contact with the component of interest during the lysis procedure. These degrading enzymes may also be present outside the cells or may have been spatially separated in different cellular compartments prior to lysis. As the lysis takes place, the component of interest becomes exposed to said degrading enzymes. Other components released during this process may e.g. be endotoxins belonging to the family of lipopolysaccharides which are toxic to cells and can cause problems for products intended to be used in human or animal therapy. There is a variety of means to tackle the above-mentioned problem. It is common to use chaotropic agents such as guanidinium thiocyanate or anionic, cationic, zwitterionic or non- ionic detergents when nucleic acids are intended to be set free. It is also an advantage to use proteases which rapidly degrade the previously described enzymes or unwanted proteins. However, this may produce another problem as said substances or enzymes can interfere with reagents or components in subsequent steps.

Enzymes which can be advantageously used in such lysis or sample preparation processes mentioned above are enzymes which cleave the amide linkages in protein substrates and which are classified as proteases, or (interchangeably) peptidases (see Walsh, 1979, Enzymatic Reaction Mechanisms. W. H. Freeman and Company, San Francisco, Chapter 3). Proteases used in the prior art comprise alkaline proteases (WO 98/04730) or acid proteases (US 5,386,024). A protease which has been widely used for sample preparation in the isolation of nucleic acids in the prior art is proteinase K from Tritirachium album (see e.g. Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) which is active around neutral pH and belongs to a family of proteases known to the person skilled in the art as subtilisins. Especially advantageous for the use in lysis or sample preparation processes mentioned above is the enzyme esperase, a robust protease that retains its activity at both high alkalinity and at high temperatures (EP 1 201 753).

In the sample preparation steps following the lysis step, the component of interest is further enriched. If the non-proteinaceous components of interest are e.g. nucleic acids, they are normally extracted from the complex lysis mixtures before they are used in a probe-based assay.

There are several methods for the purification of nucleic acids:

- sequence-dependent or biospecific methods as e.g.:

• affinity chromatography

• hybridization to immobilized probes

sequence-independent or physico-chemical methods as e.g. :

• liquid-liquid extraction with e.g. phenol-chloroform

· precipitation with e.g. pure ethanol

• extraction with filter paper

• extraction with micelle-forming agents as cetyl-trimethyl-ammonium- bromide

• binding to immobilized, intercalating dyes, e.g. acridine derivatives

• adsorption to silica gel or diatomic earths • adsorption to magnetic glass particles (MGP) or organo-silane particles under chaotropic conditions

Particularly interesting for purification purposes is the adsorption of nucleic acids to a glass surface although other surfaces are possible. Many procedures for isolating nucleic acids from their natural environment have been proposed in recent years by the use of their binding behavior to glass surfaces. If unmodified nucleic acids are the target, a direct binding of the nucleic acids to a material with a silica surface may be preferred because, among other reasons, the nucleic acids do not have to be modified, and even native nucleic acids can be bound. These processes are described in detail by various documents. In Vogelstein B. et al, Proc. Natl. Acad. USA 76 (1979) 615-9, for instance, a procedure for binding nucleic acids from agarose gels in the presence of sodium iodide to ground flint glass is proposed. The purification of plasmid DNA from bacteria on glass dust in the presence of sodium perchlorate is described in Marko M. A. et al, Anal. Biochem. 121 (1982) 382-387. In DE-A 37 34 442, the isolation of single-stranded M13 phage DNA on glass fiber filters by precipitating phage particles using acetic acid and lysis of the phage particles with perchlorate is described. The nucleic acids bound to the glass fiber filters are washed and then eluted with a methanol-containing Tris/EDTA buffer. A similar procedure for purifying DNA from lambda phages is described in Jakobi R. et al., Anal. Biochem. 175 (1988) 196-201. The procedure entails the selective binding of nucleic acids to glass surfaces in chaotropic salt solutions and separating the nucleic acids from contaminants such as agarose, proteins or cell residue. To separate the glass particles from the contaminants, the particles may be either centrifuged or fluids are drawn through glass fiber filters. This is a limiting step, however, that prevents the procedure from being used to process large quantities of samples. The use of magnetic particles to immobilize nucleic acids after precipitation by adding salt and ethanol is more advantageous and described e.g. in Alderton R. P. et al., S., Anal. Biochem. 201 (1992) 166-169 and PCT GB 91/00212. In this procedure, the nucleic acids are agglutinated along with the magnetic particles. The agglutinate is separated from the original solvent by applying a magnetic field and performing a wash step. After one wash step, the nucleic acids are dissolved in a Tris buffer. This procedure has a disadvantage, however, in that the precipitation is not selective for nucleic acids. Rather, a variety of solid and dissolved substances are agglutinated as well. As a result, this procedure cannot be used to remove significant quantities of any inhibitors of specific enzymatic reactions that may be present. Magnetic, porous glass is also commercially available that contains magnetic particles in a porous, particular glass matrix and is covered with a layer containing streptavidin. This product can be used to isolate biological materials, e.g., proteins or nucleic acids, if they are modified in a complex preparation step so that they bind covalently to biotin. Magnetizable particular adsorbents proved to be very efficient and suitable for automatic sample preparation. Ferrimagnetic and ferromagnetic as well as superparamagnetic pigments are used for this purpose. Magnetic glass particles and methods using them are described in WO 01/37291. Particularly useful for the nucleic acid isolation in the context of the disclosure is the method according to R. Boom et al. (J Clin Microbiol. 28 (1990), 495-503).

The term "solid support material" comprises any of the solid materials mentioned above in connection with the immobilization of nucleic acids, e.g. magnetic glass particles, glass fibers, glass fiber filters, filter paper etc., while the solid support material is not limited to these materials.

Thus, an aspect of the disclosure is the process described above, wherein the solid support material comprises one or more of the materials selected from silica, metal, metal oxides, plastic, polymers and nucleic acids. In an embodiment of the disclosure, the solid support material is magnetic glass particles.

"Immobilize", in the context of the disclosure, means to capture objects such as e.g. nucleic acids in a reversible or irreversible manner. Particularly, "immobilized on the solid support material", means that the object or objects are associated with the solid support material for the purpose of their separation from any surrounding media, and can be recovered e.g. by separation from the solid support material at a later point. In this context, "immobilization" can e.g. comprise the adsorption of nucleic acids to glass or other suitable surfaces of solid materials as described supra. Moreover, nucleic acids can be "immobilized" specifically by binding to capture probes, wherein nucleic acids are bound to essentially complementary nucleic acids attached to a solid support by base-pairing. In the latter case, such specific immobilization leads to the predominant binding of target nucleic acids.

After the purification or isolation of the nucleic acids including the target nucleic acids from their natural surroundings, analysis may be performed e.g. via the simultaneous amplification described supra.

"Simultaneously", in the sense of the disclosure, means that two actions, such as amplifying a first and a second or more nucleic acids, are performed at the same time and under the same physical conditions. In one embodiment, simultaneous amplification of the at least first and second target nucleic acids is performed in one vessel. In another embodiment, simultaneous amplification is performed with at least one nucleic acid in one vessel and at least a second nucleic acid in a second vessel, at the same time and under the same physical conditions, particularly with respect to temperature and incubation time wherein the internal control nucleic acid mentioned above is present each of said vessels.

The "first target nucleic acid" and the "second target nucleic acid" are different nucleic acids. A "fluid sample" is any fluid material that can be subjected to a diagnostic assay targeting nucleic acids and may be derived from a biological source. For example, said fluid sample is derived from a human and is a body liquid. In an embodiment of the disclosure, the fluid sample is human blood, urine, sputum, sweat, swab, pipettable stool, or spinal fluid.

The term "reaction vessel" comprises, but is not limited to, tubes or the wells of plates such as microwell, deepwell or other types of multiwell plates, in which a reaction for the analysis of the fluid sample such as e.g. reverse transcription or a polymerase chain reaction takes place. The outer limits or walls of such vessels are chemically inert such that they do not interfere with the analytical reaction taking place within. For example, the isolation of the nucleic acids as described above is also carried out in a multiwell plate.

In this context, multiwell plates in analytical systems allow parallel separation and analyzing or storage of multiple samples. Multiwell plates may be optimized for maximal liquid uptake, or for maximal heat transfer. A multiwell plate for use in the context of the present disclosure can be optimized for incubating or separating an analyte in an automated analyzer. For example, the multiwell plate is constructed and arranged to contact a magnetic device and/or a heating device.

Said multiwell plate, which is interchangeably termed "processing plate" in the context of the disclosure, comprises:

a top surface comprising multiple vessels with openings at the top arranged in rows. The vessels comprise an upper part, a center part and a bottom part. The upper part is joined to the top surface of the multiwell plate and comprises two longer and two shorter sides. The center part has a substantially rectangular cross-section with two longer sides and two shorter sides;

two opposing shorter and two opposing longer side walls and

a base, wherein said base comprises an opening constructed and arranged to place the multiwell plate in contact with said magnetic device and/or a heating device.

In an embodiment of the multiwell plate, adjacent vessels within one row are joined on the longer side of said almost rectangular shape.

For example, the multiwell plate comprises a continuous space which is located between adjacent rows of vessels. Said continuous space is constructed and arranged to accommodate a plate-shaped magnetic device. In an embodiment, the bottom part of the vessels comprises a spherical bottom. In an embodiment, the bottom part of said vessels comprises a conical part located between said central part and said spherical bottom.

In an embodiment, the top surface comprises ribs, wherein said ribs surround the openings of the vessels. For example, one shorter side of said upper part of the vessels comprises a recess, said recess comprising a bent surface extending from the rib to the inside of the vessel.

Furthermore, in an embodiment, the vessels comprise a rounded inside shape.

For fixation to the processing or incubation stations, the base may comprises a rim comprising recesses. Latch clips on a station of an analyzer can engage with said recesses to fix the plate on a station.

In an embodiment, the vessels comprise an essentially constant wall thickness.

For example, the processing plate (101) in the context of the present disclosure is a 1- component plate. Its top surface (110) comprises multiple vessels (103) (Fig. 5, Fig. 6). Each vessel has an opening (108) at the top and is closed at the bottom end (112). The top surface (HO) comprises ribs (104) which are may be elevated relative to the top surface (110) and surround the openings (108) of the vessels (103). This prevents contamination of the contents of the vessels (103) with droplets of liquid that may fall onto the top surface (110) of the plate (101). Views of an exemplary process plate are shown in Figs. 3 to 8.

The footprint of the processing plate (101) may comprise a length and width of the base corresponding to ANSI SBS footprint format. For example, the length is 127.76 mm +/- 0.25 mm, and the width is 85.48 mm +/- 0.25 mm. Thus, the plate (101) has two opposing shorter side walls (109) and two opposing longer side walls (118). The processing plate (101) comprises form locking elements (106) for interacting with a handler (500, Fig. 12). The processing plate (101) can be gripped, transported and positioned quickly and safely at high speed while maintaining the correct orientation and position. For example, the form locking elements (106) for gripping are located within the upper central part, for example the upper central third of the processing plate (101). This has the advantage that a potential distortion of the processing plate (101) has only a minor effect on the form locking elements (106) and that the handling of the plate (101) is more robust.

The processing plate (101) may comprise hardware -identifiers (102) and (115). The hardware identifiers (102) and (115) are unique for the processing plate (101) and different from hardware identifiers of other consumables used in the same system. The hardware identifiers (102, 115) may comprise ridges (119) and/or recesses (125) on the side walls of the consumables, wherein said pattern of ridges (119) and/or recesses (125) is unique for a specific type of consumable, for example the processing plate (101). This unique pattern is also referred to herein as a unique "surface geometry". The hardware-identifiers (102, 115) ensure that the user can only load the processing plate (101) into the appropriate stacker position of an analytical instrument in the proper orientation. On the sides of processing plate (101), guiding elements (116) and (117) are comprised (Fig. 3, Fig. 4). They prevent canting of the processing plate (101). The guiding elements (116, 117) allow the user to load the processing plates (101) with guiding elements (116, 117) as a stack into an analytical instrument which is then transferred vertically within the instrument in a stacker without canting of the plates.

The center part (120) of the vessels (103) has an almost rectangular cross section (Fig. 6, Fig. 7). They are separated along the longer side (118) of the almost rectangular shape by a common wall (113) (Fig. 3). The row of vessels (103) formed thereby has the advantage that despite the limited space available, they have a large volume, for example 4 ml. Another advantage is that because of the essentially constant wall thickness, the production is very economical. A further advantage is that the vessels (103) strengthen each other and, thus, a high stability of the shape can be obtained.

Between the rows of vessels (103), a continuous space (121) is located (Fig. 6, Fig. 7). The space (121) can accommodate magnets (202, 203) or heating devices (128) (Fig. 11). These magnets (202, 203) and heating devices (128) are for example solid devices. Thus, magnetic particles (216) comprised in liquids (215) which can be held in the vessels (103) can be separated from the liquid (215) by exerting a magnetic field on the vessels (103) when the magnets (202, 203) are brought into proximity of the vessels (103). Or the contents of the vessels (103) can be incubated at an elevated, controlled temperature when the processing plate (101) is placed on the heating device (128). Since the magnets (202, 203) or heating devices (128) can be solid, a high energy density can be achieved. The almost rectangular shape of the central part (120) of the vessels (103) (Fig. 10) also optimizes the contact between the vessel wall (109) and a flat shaped magnet (202) or heating device (128) by optimizing the contact surface between vessel (103) and magnet (202) or heating device (128) and thus enhancing energy transfer into the vessel (103).

In the area of the conical bottom (111) of the vessels, the space (121) is even more pronounced and can accommodate further magnets (203). The combination of the large magnets (202) in the upper area and the smaller magnets (203) in the conical area of the vessels allows separation of magnetic particles (216) in larger or small volumes of liquid (215). The small magnets (203), thus, make it easier to sequester the magnetic particles (216) during eluate pipetting. This makes it possible to pipette the eluate with minimal loss by reducing the dead volume of the magnetic particle (216) pellet. Furthermore, the presence of magnetic particles (216) in the transferred eluate is minimized.

At the upper end of the vessels (103), one of the shorter side walls (109) of the vessel (103) comprises an reagent inlet channel (105) which extends to the circumferential rib (104) (Figs. 3, 4, 7). The reagents are pipetted onto the reagent inlet channel (105) and drain off the channel (105) into the vessel (103). Thus, contact between the pipet needle or tip (3, 4) and liquid contained in the vessel is prevented. Furthermore, splashes resulting from liquid being directly dispensed into another liquid (215) contained in the vessels (103), which may cause contamination of the pipet needle or tip (3, 4) or neighboring vessels (103) is prevented. Sequential pipetting onto the reagent inlet channel (105) of small volumes of reagents followed by the largest volume of another reagent ensures that the reagents which are only added in small amounts are drained completely into the vessel (103). Thus, pipetting of small volumes of reagents is possible without loss of accuracy of the test to be performed.

On the inside, on the bottom of the vessels (111, 112), the shape becomes conical (111) and ends with a spherical bottom (112) (Fig. 6. Fig. 7). The inside shape of the vessel (114), including the rectangular center part (120), is rounded. The combination of spherical bottom (112), rounded inside shape (114), conical part (111) and refined surface of the vessels (103) leads to favorable fluidics which facilitate an effective separation and purification of analytes in the processing plate (101). The spherical bottom (112) allows an essentially complete use of the separated eluate and a reduction of dead-volume which reduces the carryover of reagents or sample cross-contamination.

The rim on the base (129) of the processing plate (101) comprises recesses (107) for engagement with latch clips (124) on the processing station (201) or heating device (128) or analytical instrument (126) (Fig. 5, Fig. 9). The engagement of the latch clips (124) with the recesses (107) allows positioning and fixation of the processing plate (101) on the processing station (201). The presence of the recesses (107) allows the latch force to act on the processing plate (101) almost vertically to the base (129). Thus, only small forces acting sideways can occur. This reduces the occurrence of strain, and, thus, the deformation of the processing plate (101). The vertical latch forces can also neutralize any deformations of the processing plate (101) leading to a more precise positioning of the spherical bottoms (111) within the processing station (201). In general, the precise interface between the processing plate (101) and the processing station (201) or heating device (128) within an analyzer reduces dead-volumes and also reduces the risk of sample cross-contamination. A "separation station" is a device or a component of an analytical system allowing for the isolation of the solid support material from the other material present in the fluid sample. Such a separation station can e.g. comprise, but is not limited to, a centrifuge, a rack with filter tubes, a magnet, or other suitable components. In an embodiment of the disclosure, the separation station comprises one or more magnets. For example, one or more magnets are used for the separation of magnetic particles, for example magnetic glass particles, as a solid support. If, for example, the fluid sample and the solid support material are combined together in the wells of a multiwell plate, then one or more magnets comprised by the separation station can e.g. be contacted with the fluid sample itself by introducing the magnets into the wells, or said one or more magnets can be brought close to the outer walls of the wells in order to attract the magnetic particles and subsequently separate them from the surrounding liquid.

In an embodiment, the separation station is a device that comprises a multiwell plate comprising vessels with an opening at the top surface of the multiwell plate and a closed bottom. The vessels comprise an upper part, a center part and a bottom part, wherein the upper part is joined to the top surface of the multiwell plate and may comprise two longer and two shorter sides. The center part has a substantially rectangular cross-section with two longer sides, wherein said vessels are aligned in rows. A continuous space is located between two adjacent rows for selectively contacting at least one magnet mounted on a fixture with the side walls in at least two Z-positions. The device further comprises a magnetic separation station comprising at least one fixture. The fixture comprises at least one magnet generating a magnetic field. A moving mechanism is present which vertically moves said at least one fixture comprising at least one magnet at least between first and second positions with respect to the vessels of the multiwell plate. For example, said at least two Z-positions of the vessels comprise the side walls and the bottom part of said vessels. The magnetic field of said at least one magnet may draw the magnetic particles to an inner surface of the vessel adjacent to said at least one magnet when said at least one magnet is in said first position. The effect of said magnetic field is less when said at least one magnet is in said second position than when said at least one magnet is in said first position. For example, the fixture comprising said at least one magnet comprises a frame. The vessels have features as described above in the context of multiwell plate/ processing plate. One such feature is that at least a part of said vessels has a substantially rectangular cross-section orthogonal to the axis of said vessels. In said first position, said at least one magnet is adjacent to said part of said vessels. Adjacent is understood to mean either in close proximity such as to exert a magnetic field on the contents of the vessel, or in physical contact with the vessel.

The separation station comprises a frame to receive the multiwell plate, and latch-clips to attach the multiwell plate. For example, the separation station comprises two types of magnets. This embodiment is further described below.

A second embodiment is described below, which comprises a spring which exerts a pressure on the frame comprising the magnets such that the magnets are pressed against the vessels of the multiwell plate.

The first magnets may be constructed and arranged to interact with vessels of a multiwell plate for exerting a magnetic field on a large volume of liquid comprising magnetic particles held in said vessels. Said second magnets may be constructed and arranged to interact with vessels of a multiwell plate for exerting a magnetic field on a small volume of liquid comprising magnetic particles held in said vessels. Said first and second magnets can be moved to different Z-positions.

Useful in the context of the present disclosure and said separation station is further a method of isolating and purifying a nucleic acid. The method comprises the steps of binding a nucleic acid to magnetic particles in a vessel of a multiwell plate. The vessel comprises an upper opening, a central part and a bottom part. The bound material is then separated from unbound material contained in a liquid when the major part of the liquid is located above the section where the conical part of the vessel is replaced by the central part with the rectangular shape, by moving a magnet from a second position to a first position and, in said first position, applying a magnetic field to the central part and, optionally, additionally applying a magnetic field to the bottom part of said vessel. The magnetic particles can optionally be washed with a washing solution. A small volume of liquid, wherein the major part of the liquid is located below the section where the conical part of the vessel is replaced by the central part with the rectangular shape is separated from said magnetic particles by selectively applying a magnetic field to the bottom part of said vessel.

Useful in the context of the present disclosure is also a magnetic separation station for separating a nucleic acid bound to magnetic particles, said separation station comprising first magnets which are constructed and arranged to interact with vessels of a multiwell plate for exerting a magnetic field on a large volume of liquid comprising magnetic particles held in said vessels, and second magnets constructed and arranged to interact with vessels of a multiwell plate for exerting a magnetic field on a small volume of liquid comprising magnetic particles held in said vessels, and wherein said first and second magnets can be moved to different Z-positions. Embodiments of the magnetic separation station are described herein. A first embodiment of a separation station (201) is described below. The first embodiment of said separation station (201) comprises at least two types of magnets (202, 203). The first, long type of magnet (202) is constructed and arranged to fit into the space (121) of the processing plate (101). Magnet (202), thus, exerts a magnetic field on the liquid (215) in the vessel (103) to sequester magnetic particles (216) on the inside of the vessel wall. This allows separation of the magnetic particles (216) and any material bound thereto and the liquid (215) inside the vessel (103) when a large volume of liquid (215) is present. Magnet (202) has an elongated structure and is constructed and arranged to interact with the essentially rectangular central part (120) of the vessel. Thus, magnet (202) is used when the major part of the liquid (215) is located above the section where the conical part (111) of the vessel (103) is replaced by the central part (120) with the rectangular shape. As shown in Fig. 40, the construction of the magnets (202) comprises fixtures (204, 204a) comprising magnets (202) which fit into the space (121) between the rows of vessels (103) in the processing plate (101). Another embodiment of magnets (202) comprises magnets (202) arranged on fixtures (204, 204a). The magnets (203) of the separation station (201) are smaller, and can interact with the conical part (111) of the vessel (103). This is shown in Fig. 10. Magnets (203) are arranged on a base (205) which can be moved into the space (121) of the processing plate (101). Each magnet (202, 203) is constructed to interact with two vessels (103) in two adjacent rows. In an embodiment, the processing plate (101) has 6 rows of 8 vessels (103). A separation station (201) which can interact with the processing plate (101) has three fixtures (204, 204a) comprising magnets (202) and four bases (205) comprising magnets (203). An embodiment is also included wherein the separation station has four magnetic fixtures (204, 204a) comprising magnets (202) and three magnetic bases (205) comprising magnets (203).

The magnets (202, 203) are movable. The separation station (201) comprises a mechanism to move the fixtures (204, 204a) and the bases (205). All fixtures (204, 204a) are interconnected by a base (217) and are, thus, moved coordinately. All magnets (203) are joined to one base (218) and are, thus, moved coordinately. The mechanism for moving the magnetic plates (202) and (203) is constructed and arranged to move the two types of magnetic plates (202, 203) to a total of four end positions:

In Fig. 40 a-c, the magnets (203) are located in proximity of the conical part of the vessels (103) of the processing plate (101). This is the uppermost position of magnets (203), and is the separation position. In this Figure, the magnets (202) are located in the lowermost position. They are not involved in separation when they are in this position.

In an embodiment shown in Fig. 10, the base (217) of magnets (202) is connected to a positioning wheel (206). The base (217) comprises a bottom end (207) which is flexibly in contact with a connecting element (208) by a moving element (209). Said moving element is constructed and arranged to move the connecting element (208) along a rail (212) from one side to the other. Said moving element (209) is fixed to the connecting element (208) with a pin (220). Said connecting element (208) is fixed to the positioning wheel (206) by screw

(210) . Connecting element (208) is also connected to axis (211). Said connecting element (208) is a rectangular plate. As the positioning wheel (206) moves eccentrically, around an axis (211), such that the screw (210) moves from a point above the eccentric axis to a point below the eccentric axis, moving element (209) and the bottom end (207) of the base (204) with the magnets (202) attached thereto are moved from the uppermost position to the lowermost position. The base (218) is mounted on a bottom part (219) and is connected, at its lower end, with pin (213) to a moving element (214), which can be a wheel, which interacts with the positioning wheel (206). When the positioning wheel (206) rotates around the axis

(211) , wheel (214) moves along positioning wheel (206). If the wheel (214) is located on a section of positioning wheel (206) where the distance from the axis (211) is short, the magnets (203) are in their lowermost position. When wheel (214) is located on a section of positioning wheel (206) where the distance from the axis (211) is at a maximum, the magnets (203) are in their uppermost position. Thus, in an embodiment of the first embodiment of the separation station, the location of the magnets (203) is controlled by the shape of the positioning wheel (206). When moving element (209) moves along the central, rounded upper or lower part (212a) of rail (212), the small type of magnets (203) are moved up and down. When the moving element (209) is located on the side (212b) of bottom end (207) and moves up or down, the magnets (202) are moved up- or downwards. The positioning wheel can be rotated by any motor (224).

In an embodiment, a spring (225) is attached to the base (222) of the separation station and the base (218) of magnets (203) to ensure that magnets (203) are moved into the lowermost position when they are moved downwards.

The term "pin" as used herein relates to any fixation element, including screws or pins.

In a second embodiment, the separation station (230) comprises at least one fixture (231) comprising at least one magnet (232), a number of magnets equal to a number of vessels (103) in a row (123). The separation station (230) comprises a number of fixtures (231) equal to the number of rows (123) of the multiwell plate (101) hereinbefore described. For example, six fixtures (231) are mounted on the separation station (230). At least one magnet (232) is mounted on one fixture (231). For example, the number of magnets (232) equals the number of vessels (103) in one row (123). For example, eight magnets (232) are mounted on one fixture (231). For example, one type of magnet (232) is comprised on said fixture (231). For example, the magnet (232) is mounted on one side oriented towards the vessels with which the magnet interacts.

The fixture (231) is mounted on a base (233). For example, said mount is flexible. The base (233) comprises springs (234) mounted thereon. The number of springs (234) is at least one spring per fixture (231) mounted on said base (233). The base further comprises a chamfer (236) which limits the movement of the spring and, consequently, the fixture (231) comprising the magnets (232). For example, any one of said springs (234) is constructed and arranged to interact with a fixture (231). For examples, said spring (234) is a yoke spring. Said interaction controls the horizontal movement of the fixtures (231). Furthermore, the separation station (230) comprises a frame (235). The base (233) with fixtures (231) is connected to the frame (235) by a moving mechanism as described hereinbefore for the magnets (232) of the first embodiment.

For example, said base (233) and fixture (231) is constructed and arranged to move vertically (in Z-direction).

The multiwell plate (101) hereinbefore described is inserted into the separation station (230). The fixture (231) comprising the magnets (232) is moved vertically. Any one fixture (232) is, thus, moved into a space (121) between two rows (123) of vessels (103). The vertical movement brings the magnets (232) mounted on a fixture (231) into contact with the vessels (103). The Z-position is chosen depending on the volume of liquid (215) inside the vessels (103). For large volumes, the magnets (232) contact the vessels (103) in a center position (120) where the vessels (103) are of an almost rectangular shape. For small volumes of liquid (215) where the major part of the liquid (215) is located below the center part (120) of the vessels (103), the magnets (232) contact the conical part (111) of the vessels (103).

A spring is attached to the base (233) of any one frame (231) (Fig. 9 a), b)). The spring presses the magnets (232) against the vessels (103). This ensures a contact between magnets (232) and vessels (103) during magnetic separation. For example, the magnet (232) contacts the vessel (103) on the side wall (109) located underneath the inlet (105). This has the advantage that liquid which is added by pipetting flows over the sequestered magnetic particles and ensures that particles are resuspended and that all samples in all vessels are treated identically.

This embodiment is particularly suited to separate a liquid (215) comprised in a multiwell plate (101) as hereinbefore described, from magnetic particles (216) when different levels of liquid (215) are contained in the vessels ( 103) of said multiwell plate (101).

A "wash buffer" is a fluid that is designed to remove undesired components, especially in a purification procedure. Such buffers are well known in the art. In the context of the purification of nucleic acids, the wash buffer is suited to wash the solid support material in order to separate the immobilized nucleic acid from any unwanted components. The wash buffer may, for example, contain ethanol and/ or chaotropic agents in a buffered solution or solutions with an acidic pH without ethanol and/ or chaotropic agents as described above. Often the washing solution or other solutions are provided as stock solutions which have to be diluted before use.

The washing in the process according to the disclosure requires a more or less intensive contact of the solid support material and the nucleic acids immobilized thereon with the wash buffer. Different methods are possible to achieve this, e.g. shaking the wash buffer with the solid support material in or along with the respective vessel or vessels. Another advantageous method is aspirating and dispensing the suspension comprising wash buffer and solid support material one or more times. This method may be carried out using a pipet, wherein said pipet comprises a disposable pipet tip into which said suspension is aspirated and from which it is dispensed again. Such a pipet tip can be used several times before being discarded and replaced. Disposable pipet tips may have a volume of at least 10 μΐ, or at least 15 μΐ, or at least 100 μΐ, or at least 500 μΐ, or at least 1 ml, or about 1 ml. Pipets can also be pipetting needles.

Thus, an aspect of the disclosure is the process described above, wherein said washing in step d. comprises aspirating and dispensing the wash buffer comprising the solid support material. For downstream processing of the isolated nucleic acids, it can be advantageous to separate them from the solid support material before subjecting them to amplification.

Therefore, an aspect of the disclosure is the process described above, wherein said process further comprises in step d. the step of eluting the purified nucleic acids from the solid support material with an elution buffer after washing said solid support material.

An "elution buffer" in the context of the disclosure is a suitable liquid for separating the nucleic acids from the solid support. Such a liquid may e.g. be distilled water or aqueous salt solutions, such as e.g. Tris buffers like Tris HC1, or HEPES, or other suitable buffers known to the skilled artisan. The pH value of such an elution buffer can be alkaline or neutral. Said elution buffer may contain further components such as e.g. chelators like EDTA, which stabilizes the isolated nucleic acids by inactivation of degrading enzymes.

The elution can be carried out at elevated temperatures, such that an embodiment of the disclosure is the process described above, wherein step d. is carried out at a temperature between 70°C and 90°C, or at a temperature of 80°C.

"Amplification reagents", in the context of the disclosure, are chemical or biochemical components that enable the amplification of nucleic acids. Such reagents comprise, but are not limited to, nucleic acid polymerases, buffers, mononucleotides such as nucleoside triphosphates, oligonucleotides e.g. as oligonucleotide primers, salts and their respective solutions, detection probes, dyes, and more.

As is known in the art, a "nucleoside" is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines.

"Nucleotides" are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2'-, 3'- or 5'-hydroxyl moiety of the sugar. A nucleotide is the monomeric unit of an "oligonucleotide", which can be more generally denoted as an "oligomeric compound", or a "polynucleotide", more generally denoted as a "polymeric compound". Another general expression for the aforementioned is deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

According to the disclosure, an "oligomeric compound" is a compound consisting of "monomeric units" which may be nucleotides alone or non-natural compounds (see below), more specifically modified nucleotides (or nucleotide analogs) or non-nucleotide compounds, alone or combinations thereof.

"Oligonucleotides" and "modified oligonucleotides" (or "oligonucleotide analogs") are subgroups of oligomeric compounds. In the context of this disclosure, the term "oligonucleotide" refers to components formed from a plurality of nucleotides as their monomeric units. The phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage. Oligonucleotides and modified oligonucleotides (see below) may be synthesized as principally described in the art and known to the expert in the field. Methods for preparing oligomeric compounds of specific sequences are known in the art, and include, for example, cloning and restriction of appropriate sequences and direct chemical synthesis. Chemical synthesis methods may include, for example, the phosphotriester method described by Narang S. A. et al, Methods in Enzymology 68 (1979) 90-98, the phosphodiester method disclosed by Brown E. L., et al, Methods in Enzymology 68 (1979) 109-151, the phosphoramidite method disclosed in Beaucage et al, Tetrahedron Letters 22 (1981) 1859, the H-phosphonate method disclosed in Garegg et al, Chem. Scr. 25 (1985) 280-282 and the solid support method disclosed in US 4,458,066.

In the process described above, the oligonucleotides may be chemically modified, i.e. the primer and/ or the probe comprise a modified nucleotide or a non-nucleotide compound. The probe or the primer is then a modified oligonucleotide.

"Modified nucleotides" (or "nucleotide analogs") differ from a natural nucleotide by some modification but still consist of a base, a pentofuranosyl sugar, a phosphate portion, base-like, pentofuranosyl sugar-like and phosphate-like portion or combinations thereof. For example, a label may be attached to the base portion of a nucleotide whereby a modified nucleotide is obtained. A natural base in a nucleotide may also be replaced by e.g. a 7-deazapurine whereby a modified nucleotide is obtained as well.

A "modified oligonucleotide" (or "oligonucleotide analog"), belonging to another specific subgroup of oligomeric compounds, possesses one or more nucleotides and one or more modified nucleotides as monomeric units. Thus, the term "modified oligonucleotide" (or "oligonucleotide analog") refers to structures that function in a manner substantially similar to oligonucleotides and can be used interchangeably. From a synthetical point of view, a modified oligonucleotide (or an oligonucleotide analog) can for example be made by chemical modification of oligonucleotides by appropriate modification of the phosphate backbone, ribose unit or the nucleotide bases (Uhlmann and Peyman, Chemical Reviews 90 (1990) 543; Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134). Representative modifications include phosphorothioate, phosphorodithioate, methyl phosphonate, phosphotriester or phosphoramidate inter-nucleoside linkages in place of phosphodiester internucleoside linkages; deaza- or azapurines and -pyrimidines in place of natural purine and pyrimidine bases, pyrimidine bases having substituent groups at the 5 or 6 position; purine bases having altered substituent groups at the 2, 6 or 8 positions or 7 position as 7-deazapurines; bases carrying alkyl-, alkenyl-, alkinyl or aryl-moieties, e.g. lower alkyl groups such as methyl, ethyl, propyl, butyl, tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, or aryl groups like phenyl, benzyl, naphtyl; sugars having substituent groups at, for example, their 2' position; or carbocyclic or acyclic sugar analogs. Other modifications are known to those skilled in the art. Such modified oligonucleotides (or oligonucleotide analogs) are best described as being functionally interchangeable with, yet structurally different from, natural oligonucleotides. In more detail, exemplary modifications are disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134 or WO 02/12263. In addition, modification can be made wherein nucleoside units are joined via groups that substitute for the internucleoside phosphate or sugar phosphate linkages. Such linkages include those disclosed in Verma S., and Eckstein F., Annu. Rev. Biochem. 67 (1998) 99-134. When other than phosphate linkages are utilized to link the nucleoside units, such structures have also been described as "oligonucleosides".

A "nucleic acid" as well as the "target nucleic acid" is a polymeric compound of nucleotides as known to the expert skilled in the art. "Target nucleic acid" is used herein to denote a nucleic acid in a sample which should be analyzed, i.e. the presence, non-presence and/or amount thereof in a sample should be determined.

The term "primer" is used herein as known to the expert skilled in the art and refers to oligomeric compounds, primarily to oligonucleotides, but also to modified oligonucleotides that are able to prime DNA synthesis by a template-dependent DNA polymerase, i.e. the 3 '- end of the e.g. primer provides a free 3'-OH group whereto further nucleotides may be attached by a template-dependent DNA polymerase establishing 3'- to 5'-phosphodiester linkage whereby deoxynucleoside triphosphates are used and whereby pyrophosphate is released.

A "probe" also denotes a natural or modified oligonucleotide. As known in the art, a probe serves the purpose to detect an analyte or amplificate. In the case of the process described above, probes can be used to detect the amplificates of the target nucleic acids. For this purpose, probes typically carry labels.

"Labels", often referred to as "reporter groups", are generally groups that make a nucleic acid, in particular oligonucleotides or modified oligonucleotides, as well as any nucleic acids bound thereto distinguishable from the remainder of the sample (nucleic acids having attached a label can also be termed labeled nucleic acid binding compounds, labeled probes or just probes). Exemplary labels are fluorescent labels, which are e.g. fluorescent dyes such as a fluorescein dye, a rhodamine dye, a cyanine dye, and a coumarin dye. Exemplary fluorescent dyes are FAM, HEX, JA270, CAL635, Coumarin343, Quasar705, Cyan500, CY5.5, LC-Red 640, LC-Red 705.

In the context of the disclosure, any primer and/or probe may be chemically modified, i.e. the primer and/ or the probe comprise a modified nucleotide or a non-nucleotide compound. The probe or the primer is then a modified oligonucleotide. A method of nucleic acid amplification is the Polymerase Chain Reaction (PCR) which is disclosed, among other references, in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, and 4,965,188. PCR typically employs two or more oligonucleotide primers that bind to a selected nucleic acid template (e.g. DNA or RNA). Primers useful for nucleic acid analysis include oligonucleotides capable of acting as a point of initiation of nucleic acid synthesis within the nucleic acid sequences of the target nucleic acids. A primer can be purified from a restriction digest by conventional methods, or it can be produced synthetically. The primer can be single- stranded for maximum efficiency in amplification, but the primer can be double-stranded. Double-stranded primers are first denatured, i.e., treated to separate the strands. One method of denaturing double stranded nucleic acids is by heating. A "thermostable polymerase" is a polymerase enzyme that is heat stable, i.e., it is an enzyme that catalyzes the formation of primer extension products complementary to a template and does not irreversibly denature when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded template nucleic acids. Generally, the synthesis is initiated at the 3' end of each primer and proceeds in the 5' to 3' direction along the template strand. Thermostable polymerases have e.g. been isolated from Thermus flavus, T. ruber, T. thermophilus, T. aquaticus, T. lacteus, T. rubens, Bacillus stearothermophilus, and Methanothermus fervidus. Nonetheless, polymerases that are not thermostable also can be employed in PCR assays provided the enzyme is replenished.

If the template nucleic acid is double-stranded, it is necessary to separate the two strands before it can be used as a template in PCR. Strand separation can be accomplished by any suitable denaturing method including physical, chemical or enzymatic means. One method of separating the nucleic acid strands involves heating the nucleic acid until it is predominately denatured (e.g., greater than 50%, 60%, 70%, 80%, 90% or 95% denatured). The heating conditions necessary for denaturing template nucleic acid will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90°C to about 105°C for a time depending on features of the reaction such as temperature and the nucleic acid length. Denaturation is typically performed for about 5 sec to 9 min. In order to not expose the respective polymerase like e.g. the Z05 DNA Polymerase to such high temperatures for too long and thus risking a loss of functional enzyme, it can be preferred to use short denaturation steps.

In an embodiment of the disclosure, the denaturation step is up to 30 sec, or up to 20 sec, or up to 10 sec, or up to 5 sec, or about 5 sec. If the double-stranded template nucleic acid is denatured by heat, the reaction mixture is allowed to cool to a temperature that promotes annealing of each primer to its target sequence on the target nucleic acids.

The temperature for annealing can be from about 35°C to about 70°C, or about 45°C to about 65°C; or about 50°C to about 60°C, or about 55°C to about 58°C. Annealing times can be from about 10 sec to about 1 min (e.g., about 20 sec to about 50 sec; about 30 sec to about 40 sec). In this context, it can be advantageous to use different annealing temperatures in order to increase the inclusivity of the respective assay. In brief, this means that at relatively low annealing temperatures, primers may also bind to targets having single mismatches, so variants of certain sequences can also be amplified. This can be desirable if e.g. a certain organism has known or unknown genetic variants which should also be detected. On the other hand, relatively high annealing temperatures bear the advantage of providing higher specificity, since towards higher temperatures the probability of primer binding to not exactly matching target sequences continuously decreases. In order to benefit from both phenomena, in some embodiments of the disclosure the process described above comprises annealing at different temperatures, for example first at a lower, then at a higher temperature. If, e.g., a first incubation takes place at 55°C for about 5 cycles, non-exactly matching target sequences may be (pre-)amplified. This can be followed e.g. by about 45 cycles at 58°C, providing for higher specificity throughout the major part of the experiment. This way, potentially important genetic variants are not missed, while the specificity remains relatively high.

The reaction mixture is then adjusted to a temperature at which the activity of the polymerase is promoted or optimized, i.e., a temperature suitable for extension to occur from the annealed primer to generate products complementary to the nucleic acid to be analyzed. The temperature should be suitable to synthesize an extension product from each primer that is annealed to a nucleic acid template, but should not be so high as to denature an extension product from its complementary template (e.g., the temperature for extension generally ranges from about 40° to 80°C (e.g., about 50°C to about 70°C; about 60°C). Extension times can be from about 10 sec to about 5 min, or about 15 sec to 2 min, or about 20 sec to about 1 min, or about 25 sec to about 35 sec. The newly synthesized strands form a double-stranded molecule that can be used in the succeeding steps of the reaction. The steps of strand separation, annealing, and elongation can be repeated as often as needed to produce the desired quantity of amplification products corresponding to the target nucleic acids. The limiting factors in the reaction are the amounts of primers, thermostable enzyme, and nucleoside triphosphates present in the reaction. The cycling steps (i.e., denaturation, annealing, and extension) can be repeated at least once. For use in detection, the number of cycling steps will depend, e.g., on the nature of the sample. If the sample is a complex mixture of nucleic acids, more cycling steps will be required to amplify the target sequence suitable for detection. Generally, the cycling steps are repeated at least about 20 times, but may be repeated as many as 40, 60, or even 100 times.

A PCR can be carried out in which the steps of annealing and extension are performed in the same step (one-step PCR) or, as described above, in separate steps (two-step PCR). Performing annealing and extension together and thus under the same physical and chemical conditions, with a suitable enzyme such as, for example, the Z05 DNA polymerase, bears the advantage of saving the time for an additional step in each cycle, and also abolishing the need for an additional temperature adjustment between annealing and extension. Thus, the one-step PCR reduces the overall complexity of the respective assay.

In general, shorter times for the overall amplification can be preferred, as the time-to-result is reduced and leads to a possible earlier diagnosis.

Other nucleic acid amplification methods to be used comprise the Ligase Chain Reaction

(LCR; Wu D. Y. and Wallace R. B., Genomics 4 (1989) 560-69; and Barany F., Proc. Natl.

Acad. Sci. USA 88 (1991)189-193); Polymerase Ligase Chain Reaction (Barany F., PCR

Methods and Applic. 1 (1991) 5-16); Gap-LCR (WO 90/01069); Repair Chain Reaction (EP

0439182 A2), 3SR (Kwoh D.Y. et al, Proc. Natl. Acad. Sci. USA 86 (1989) 1173-1177; GuateUi J.C., et al, Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; WO 92/08808), and

NASBA (US 5,130,238). Further, there are strand displacement amplification (SDA), transcription mediated amplification (TMA), and Qb-amplification (for a review see e.g.

Whelen A. C. and Persing D. H., Annu. Rev. Microbiol. 50(1996) 349-373; Abramson R. D. and Myers T. W., Curr Opin Biotechnol 4 (1993) 41-47).

The internal control nucleic acid may exhibit the following properties relating to its sequence:

- a melting temperature from 55°C to 90°C, or from 65°C to 85°C, or from 70°C to 80°C, or about 75°C

a length of up to 500 bases or base pairs, or from 50 to 300 bases or base pairs, or from 100 to 200 bases or base pairs, or about 180 bases or base pairs

- a GC content from 30% to 70%, or from 40% to 60%, or about 50%.

In the context of the disclosure, a "sequence" is the primary structure of a nucleic acid, i.e. the specific arrangement of the single nucleobases of which the respective nucleic acids consists. It has to be understood that the term "sequence" does not denote a specific type of nucleic acid such as RNA or DNA, but applies to both as well as to other types of nucleic acids such as e.g. PNA or others. Where nucleobases correspond to each other, particularly in the case of uracil (present in RNA) and thymine (present in DNA), these bases can be considered equivalent between RNA and DNA sequences, as well-known in the pertinent art.

Clinically relevant nucleic acids are often DNA which can be derived e.g. from DNA viruses like e.g. Hepatitis B Virus (HBV), Cytomegalovirus (CMV) and others, or bacteria like e.g. Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG) and others. In such cases, it can be advantageous to use an internal control nucleic acid consisting of DNA, in order to reflect the target nucleic acids properties.

Therefore, an aspect of the disclosure is the method described above, wherein said internal control nucleic acid is DNA.

Internal control DNA can be provided as an armored particle. Armored DNA is useful as internal control nucleic acid. In a further embodiment, DNA control nucleic acids are armored using lambda phage GT11. Therefore, an aspect of the disclosure is the method described above, wherein said internal control nucleic acid is an armored nucleic acid.

A "polymerase with reverse transcriptase activity" is a nucleic acid polymerase capable of synthesizing DNA based on an RNA template. In an embodiment of the disclosure, the polymerase with reverse transcriptase activity is thermostable.

The process according to the disclosure comprises incubating a sample containing an RNA nucleic acid with an oligonucleotide primer sufficiently complementary to said RNA nucleic acid to hybridize with the latter, and a thermostable DNA polymerase in the presence of at least all four natural or modified deoxyribonucleoside triphosphates, in an appropriate buffer comprising a metal ion buffer which, in an embodiment, buffers both the pH and the metal ion concentration. This incubation is performed at a temperature suitable for said primer to hybridize to said RNA template and said DNA polymerase to catalyze the polymerization of said deoxyribonucleoside triphosphates to form a cDNA sequence complementary to the sequence of said RNA template.

As used herein, the term "cDNA" refers to a complementary DNA molecule synthesized using a ribonucleic acid strand (RNA) as a template. The RNA may e.g. be mRNA, tRNA, rRNA, or another form of RNA, such as viral RNA. The cDNA may be single-stranded, double-stranded or may be hydrogen-bonded to a complementary RNA molecule as in an RNA/cDNA hybrid.

A primer suitable for annealing to an RNA nucleic acid may also be suitable for amplification by PCR. For PCR, a second primer, complementary to the reverse transcribed cDNA strand, provides an initiation site for the synthesis of an extension product. Thermostable DNA polymerases can be used in a coupled, one-enzyme reverse transcription/amplification reaction. The term "homogeneous", in this context, refers to a two- step single addition reaction for reverse transcription and amplification of an RNA target. By homogeneous it is meant that following the reverse transcription (RT) step, there is no need to open the reaction vessel or otherwise adjust reaction components prior to the amplification step. In a non-homogeneous RT/PCR reaction, following reverse transcription and prior to amplification one or more of the reaction components such as the amplification reagents are e.g. adjusted, added, or diluted, for which the reaction vessel has to be opened, or at least its contents have to be manipulated. Both homogeneous and non-homogeneous embodiments are comprised by the scope of the disclosure.

Therefore, an aspect of the disclosure is the process described above, wherein said incubation of the polymerase with reverse transcriptase activity is carried out at different temperatures from 30°C to 75°C, or from 45°C to 70°C, or from 55°C to 65°C.

Thus, an aspect of the disclosure is the process described above, wherein the period of time for incubation of the polymerase with reverse transcriptase activity is up to 30 minutes, 20 minutes, 15 minutes, 12.5 minutes, 10 minutes, 5 minutes, or 1 minute.

A further aspect of the disclosure is the process described above, wherein the polymerase with reverse transcriptase activity and comprising a mutation is selected from the group consisting of

a) a CS5 DNA polymerase

b) a CS6 DNA polymerase

c) a Thermotoga maritima DNA polymerase

d) a Thermus aquaticus DNA polymerase

e) a Thermus thermophilus DNA polymerase

f) a Thermus flavus DNA polymerase

g) a Thermus filiformis DNA polymerase

h) a Thermus sp. spsl7 DNA polymerase

i) a Thermus sp. Z05 DNA polymerase

j) a Thermotoga neapolitana DNA polymerase

k) a Termosipho africanus DNA polymerase

1) a Thermus caldophilus DNA polymerase Particularly suitable for these requirements are enzymes carrying a mutation in the polymerase domain that enhances their reverse transcription efficiency in terms of a faster extension rate.

Therefore, an aspect of the disclosure is the process described above, wherein the polymerase with reverse transcriptase activity is a polymerase comprising a mutation conferring an improved nucleic acid extension rate and/or an improved reverse transcriptase activity relative to the respective wildtype polymerase.

In an embodiment, in the process described above, the polymerase with reverse transcriptase activity is a polymerase comprising a mutation conferring an improved reverse transcriptase activity relative to the respective wildtype polymerase.

Polymerases carrying point mutations that render them particularly useful in the context of the disclosure are disclosed in WO 2008/046612. In particular, polymerases to be can be mutated DNA polymerases comprising at least the following motif in the polymerase domain:

T-G-R-L-S-S-Xb7-Xb8-P-N-L-Q-N; wherein X _b7 is an amino acid selected from S or T and wherein X _b g is an amino acid selected from G, T, R, K, or L, wherein the polymerase comprises 3 '-5 ' exonuclease activity and has an improved nucleic acid extension rate and/or an improved reverse transcription efficiency relative to the wildtype DNA polymerase, wherein in said wildtype DNA polymerase Xb8 is an amino acid selected from D, E or N. One example is mutants of the thermostable DNA polymerase from Thermus species Z05 (described e.g. in US 5,455, 170), said variations comprising mutations in the polymerase domain as compared with the respective wildtype enzyme Z05. An embodiment for the method according to the disclosure is a mutant Z05 DNA polymerase wherein the amino acid at position 580 is selected from the group consisting of G, T, R, K and L.

For reverse transcription using a thermostable polymerase, Mn2+ can be the divalent cation and is typically included as a salt, for example, manganese chloride (MnC12), manganese acetate (Mn(OAc)2), or manganese sulfate (MnS04). If MnC12 is included in a reaction containing 50 mM Tricine buffer, for example, the MnC12 is generally present at a concentration of 0.5-7.0 mM; 0.8-1.4 mM is preferred when 200 mM of each dGTP, dATP, dUTP, and, dCTP are utilized; and 2.5-3.5 mM MnC12 is most preferred. Further, also Mg2+ may be used as a divalent cation for reverse transcription.

An "organism", as used herein, means any living single- or multicellular life form. As used herein, a virus is an organism.

Especially due to an appropriate temperature optimum, enzymes like Tth polymerase or, for example, the mutant Z05 DNA polymerase mentioned above are suited to carry out the subsequent step of amplification of the target nucleic acids. Exploiting the same enzyme for both reverse transcription an amplification contributes to the ease of carrying out the process and facilitates its automation, since the fluid sample does not have to be manipulated between the RT and the amplification step.

Therefore, in the process described above the same polymerase with reverse transcriptase activity is used for reverse transcription and for the amplification in step g. For example, the enzyme is the mutant Z05 DNA polymerase described supra.

In order not to expose the polymerase or other components of the reaction mixture to elevated temperatures for times longer than necessary, in an embodiment, steps above 90°C are up to 20 sec, or up to 15 sec, or up to 10 sec, or up to 5 sec or 5 sec long. This also reduces the time-to-result and cuts down the overall required time of the assay.

In such a homogeneous setup, it can be of considerable advantage to seal the reaction vessels prior to initiating the RT and the amplification, thereby reducing the risk of contamination. Sealing can be e.g. achieved by applying a foil that can be transparent, a cap, or by oil added to the reaction vessels and forming a lipophilic phase as a sealing layer at the top of the fluid. Thus, an aspect of the disclosure is the process described above, further comprising after step e. the step of sealing the at least two reaction vessels.

For the ease of handling and to facilitate automation, the at least two reaction vessels can be combined in an integral arrangement, so they can be manipulated together.

Consequently, an aspect of the disclosure is the process described above, wherein the at least two reaction vessels are combined in the same integral arrangement.

Integral arrangements can e.g. be vials or tubes reversibly or irreversibly attached to each other or arranged in a rack. For example, the integral arrangement is a multiwell plate.

Since nucleic acid amplification, especially but not only in the case of PCR, is very efficient if carried out as a cycling reaction, an aspect of the disclosure is the process described above, wherein the amplification reaction in step g. consists of multiple cycling steps.

Suitable nucleic acid detection methods are known to the expert in the field and are described in standard textbooks as Sambrook J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989 and Ausubel F. et al: Current Protocols in Molecular Biology 1987, J. Wiley and Sons, NY. There may be also further purification steps before the nucleic acid detection step is carried out as e.g. a precipitation step. The detection methods may include but are not limited to the binding or intercalating of specific dyes as ethidium bromide which intercalates into the double-stranded DNA and changes its fluorescence thereafter. The purified nucleic acid may also be separated by electrophoretic methods optionally after a restriction digest and visualized thereafter. There are also probe-based assays which exploit the oligonucleotide hybridization to specific sequences and subsequent detection of the hybrid.

The amplified target nucleic acids can be detected during or after the amplification reaction in order to evaluate the result of the analysis. Particularly for detection in real time, it is advantageous to use nucleic acid probes.

Thus, an aspect of the invention is the process described above, wherein a cycling step comprises an amplification step and a hybridization step, said hybridization step comprising hybridizing the amplified nucleic acids with probes.

It can be favorable to monitor the amplification reaction in real time, i.e. to detect the target nucleic acids and/or their amplificates during the amplification itself.

Therefore, an aspect of the disclosure is the process described above, wherein the probes are labeled with a donor fluorescent moiety and a corresponding acceptor fluorescent moiety. The methods set out above can be based on Fluorescence Resonance Energy Transfer (FRET) between a donor fluorescent moiety and an acceptor fluorescent moiety. A representative donor fluorescent moiety is fluorescein, and representative corresponding acceptor fluorescent moieties include LC-Red 640, LC-Red 705, Cy5, and Cy5.5. Typically, detection includes exciting the sample at a wavelength absorbed by the donor fluorescent moiety and visualizing and/or measuring the wavelength emitted by the corresponding acceptor fluorescent moiety. In the process according to the disclosure, detection can be followed by quantitating the FRET. For example, detection is performed after each cycling step. For example, detection is performed in real time. By using commercially available real-time PCR instrumentation (e.g., LightCycler™ or TaqMan®), PCR amplification and detection of the amplification product can be combined in a single closed cuvette with dramatically reduced cycling time. Since detection occurs concurrently with amplification, the real-time PCR methods obviate the need for manipulation of the amplification product, and diminish the risk of cross-contamination between amplification products. Real-time PCR greatly reduces turn-around time and is an attractive alternative to conventional PCR techniques in the clinical laboratory.

The following patent applications describe real-time PCR as used in the LightCycler™ technology: WO 97/46707, WO 97/46714 and WO 97/46712. The LightCycler™ instrument is a rapid thermal cycler combined with a microvolume fluorometer utilizing high quality optics. This rapid thermocycling technique uses thin glass cuvettes as reaction vessels. Heating and cooling of the reaction chamber are controlled by alternating heated and ambient air. Due to the low mass of air and the high ratio of surface area to volume of the cuvettes, very rapid temperature exchange rates can be achieved within the thermal chamber.

TaqMan® technology utilizes a single-stranded hybridization probe labeled with two fluorescent moieties. When a first fluorescent moiety is excited with light of a suitable wavelength, the absorbed energy is transferred to a second fluorescent moiety according to the principles of FRET. The second fluorescent moiety is generally a quencher molecule. Typical fluorescent dyes used in this format are for example, among others, FAM, HEX, CY5, JA270, Cyan and CY5.5. During the annealing step of the PCR reaction, the labeled hybridization probe binds to the target nucleic acid (i.e., the amplification product) and is degraded by the 5' to 3' exonuclease activity of the Taq or another suitable polymerase as known by the skilled artisan, such as a mutant Z05 polymerase, during the subsequent elongation phase. As a result, the excited fluorescent moiety and the quencher moiety become spatially separated from one another. As a consequence, upon excitation of the first fluorescent moiety in the absence of the quencher, the fluorescence emission from the first fluorescent moiety can be detected.

In both detection formats described above, the intensity of the emitted signal can be correlated with the number of original target nucleic acid molecules.

As an alternative to FRET, an amplification product can be detected using a double-stranded DNA binding dye such as a fluorescent DNA binding dye (e.g., SYBRGREEN I® or SYBRGOLD® (Molecular Probes)). Upon interaction with the double-stranded nucleic acid, such fluorescent DNA binding dyes emit a fluorescence signal after excitation with light at a suitable wavelength. A double-stranded DNA binding dye such as a nucleic acid intercalating dye also can be used. When double-stranded DNA binding dyes are used, a melting curve analysis is usually performed for confirmation of the presence of the amplification product. Molecular beacons in conjunction with FRET can also be used to detect the presence of an amplification product using the real-time PCR methods of the disclosure. Molecular beacon technology uses a hybridization probe labeled with a first fluorescent moiety and a second fluorescent moiety. The second fluorescent moiety is generally a quencher, and the fluorescent labels are typically located at each end of the probe. Molecular beacon technology uses a probe oligonucleotide having sequences that permit secondary structure formation (e.g. a hairpin). As a result of secondary structure formation within the probe, both fluorescent moieties are in spatial proximity when the probe is in solution. After hybridization to the amplification products, the secondary structure of the probe is disrupted and the fluorescent moieties become separated from one another such that after excitation with light of a suitable wavelength, the emission of the first fluorescent moiety can be detected.

Thus, in a method according to the disclosure is the method described above using FRET, wherein said probes comprise a nucleic acid sequence that permits secondary structure formation, wherein said secondary structure formation results in spatial proximity between said first and second fluorescent moiety.

Efficient FRET can only take place when the fluorescent moieties are in direct local proximity and when the emission spectrum of the donor fluorescent moiety overlaps with the absorption spectrum of the acceptor fluorescent moiety.

Thus, in an embodiment, said donor and acceptor fluorescent moieties are within no more than 5 nucleotides of each other on said probe.

In a further embodiment, said acceptor fluorescent moiety is a quencher.

As described above, in the TaqMan format, during the annealing step of the PCR reaction, the labeled hybridization probe binds to the target nucleic acid (i.e., the amplification product) and is degraded by the 5 '-to 3'-exonuclease activity of the Taq or another suitable polymerase as known by the skilled artisan, such as a mutant Z05 polymerase, during the subsequent elongation phase.

Thus, in an embodiment, in the process described above, amplification employs a polymerase enzyme having 5 '-to 3'-exonuclease activity.

It is further advantageous to carefully select the length of the amplicon that is yielded as a result of the process described above. Generally, relatively short amplicons increase the efficiency of the amplification reaction. Thus, an aspect of the disclosure is the process described above, wherein the amplified fragments comprise up to 450 bases, up to 300 bases, up to 200 bases, or up to 150 bases.

The internal control nucleic acid used in the present disclosure can serve as a "quantitative standard nucleic acid" which is apt to be and used as a reference in order to quantify, i.e. to determine the quantity of the target nucleic acids. For this purpose, one or more quantitative standard nucleic acids undergo all possible sample preparation steps along with the target nucleic acids. Moreover, a quantitative standard nucleic acid is processed throughout the method within the same reaction mixture. It must generate, directly or indirectly, a detectable signal both in the presence or absence of the target nucleic acid. For this purpose, the concentration of the quantitative standard nucleic acid has to be carefully optimized in each test in order not to interfere with sensitivity but in order to generate a detectable signal also e.g. at very high target concentrations. In terms of the limit of detection (LOD, see below) of the respective assay, the concentration range for the "quantitative standard nucleic acid" is 20- 5000x LOD, 20-lOOOx LOD, or 20-5000x LOD. The final concentration of the quantitative standard nucleic acid in the reaction mixture is dependent on the quantitative measuring range accomplished.

"Limit of detection" or "LOD" means the lowest detectable amount or concentration of a nucleic acid in a sample. A low "LOD" corresponds to high sensitivity and vice versa. The "LOD" is usually expressed either by means of the unit "cp/ml", particularly if the nucleic acid is a viral nucleic acid, or as IU/ml. "Cp/ml" means "copies per milliliter" wherein a "copy" is copy of the respective nucleic acid. IU/ml stands for "International units/ml", referring to the WHO standard.

A widely used method for calculating an LOD is "Probit Analysis", which is a method of analyzing the relationship between a stimulus (dose) and the quantal (all or nothing) response. In a typical quantal response experiment, groups of animals are given different doses of a drug. The percent dying at each dose level is recorded. These data may then be analyzed using Probit Analysis. The Probit Model assumes that the percent response is related to the log dose as the cumulative normal distribution. That is, the log doses may be used as variables to read the percent dying from the cumulative normal. Using the normal distribution, rather than other probability distributions, influences the predicted response rate at the high and low ends of possible doses, but has little influence near the middle.

The Probit Analysis can be applied at distinct "hitrates". As known in the art, a "hitrate" is commonly expressed in percent [%] and indicates the percentage of positive results at a specific concentration of an analyte. Thus for example, an LOD can be determined at 95% hitrate, which means that the LOD is calculated for a setting in which 95% of the valid results are positive.

In an embodiment, the process described above provides an LOD of 1 to 100 cp/ml or 0.5 to 50 IU/ml, or 1 to 75 cp/ml or 0.5 to 30 IU/ml, or 1 to 25 cp/ml or 1 to 20 IU/ml.

With respect to some examples of possible target nucleic acids from certain viruses, the process described above provides the following LODs:

• HIV: up to 60 cp/ml, up to 50 cp/ml, up to 40 cp/ml, up to 30 cp/ml, up to 20 cp/ml, or up to 15 cp/ml

• HBV: up to 10 IU/ml, up to 7.5 IU/ml, or up to 5 IU/ml

• HCV: up to 10 IU/ml, up to 7.5 IU/ml, or up to 5 IU/ml

• WNV I: up to 20 cp/ml, up to 15 cp/ml, or up to 10 cp/ml • WNV II: up to 20 cp/ml, up to 15 cp/ml, up to 10 cp/ml, or up to 5 cp/ml

• JEV: up to 100 cp/ml, up to 75 cp/ml, up to 50 cp/ml, or up to 30 cp/ml

• SLEV: up to 100 cp/ml, up to 75 cp/ml, up to 50 cp/ml, up to 25 cp/ml, or up to 10 cp/ml.

An example of how to perform calculation of quantitative results in the TaqMan format based on an internal control nucleic acid serving as a quantitative standard nucleic acid is described in the following: A titer is calculated from input data of instrument-corrected fluorescence values from an entire PCR run. A set of samples containing a target nucleic acid and an internal control nucleic acid serving as a quantitative standard nucleic acid undergo PCR on a thermocycler using a specified temperature profile. At selected temperatures and times during the PCR profile samples are illuminated by filtered light and the filtered fluorescence data are collected for each sample for the target nucleic acid and the internal control nucleic acid. After a PCR run is complete, the fluorescence readings are processed to yield one set of dye concentration data for the internal control nucleic acid and one set of dye concentration data for the target nucleic acid. Each set of dye concentration data is processed in the same manner. After several plausibility checks, the elbow values (CT) are calculated for the internal control nucleic acid and the target nucleic acid. The elbow value is defined as the point where the fluorescence of the target nucleic acid or the internal control nucleic acid crosses a predefined threshold (fluorescence concentration). Titer determination is based on the assumptions that the target nucleic acid and the internal control nucleic acid are amplified with the same efficiency and that at the calculated elbow value equal amounts of amplicon copies of target nucleic acid and internal control nucleic acid are amplified and detected. Therefore, the (CTQS - CTtarget) is linear to log (target cone / QS cone). In this context, QS denotes the internal control nucleic acid serving as a quantitative standard nucleic acid. The titer T can then be calculated for instance by using a polynomial calibration formula as in the following equation:

T = 10 (a(CTQS - CTtarget)2 + b(CTQS - CTtarget) + c)

The polynomial constants and the concentration of the quantitative standard nucleic acid are known, therefore the only variable in the equation is the difference (CTQS - CTtarget).

Further, the internal control nucleic acid can serve as a "qualitative internal control nucleic acid". A "qualitative internal control nucleic acid" is particularly useful for confirming the validity of the test result of a qualitative detection assay: Even in the case of a negative result, the qualitative internal control must be detected, otherwise the test itself is considered to be inoperative. However, in a qualitative setup, it does not necessarily have to be detected in case of a positive result. As a consequence, its concentration must be relatively low. It has to be carefully adapted to the respective assay and its sensitivity. For example, the concentration range for the qualitative internal nucleic acid, i.e. the second control nucleic acid, will comprise a range of 1 copy per reaction to 1000 copies per reaction. In relation to the respective assay's limit of detection (LOD), its concentration is between the LOD of an assay and the 25fold value of the LOD, or between the LOD and lOx LOD. Or, it is between 2x and lOx LOD. Or, it is between 5x and lOx LOD. Or, it is 5x or lOx LOD.

Herein, the internal control nucleic acid is not restricted to a particular sequence. It can be advantageous to add different internal control nucleic acids to a fluid samples, but to use only one of them for amplification e.g. by adding only primers for one of said internal control nucleic acids. In such embodiments, the internal control nucleic acid to be amplified in a certain experiment can be chosen by the person skilled in the art, thus increasing flexibility of the analysis to be carried out. In particularly advantageous embodiments, said different internal control nucleic acids can be comprised by a single nucleic acid construct, e.g. a plasmid or a different suitable nucleic acid molecule.

Therefore, an aspect of the disclosure is the process described above, wherein more than one internal control nucleic acid is added in step a., but only one of said internal control nucleic acids is amplified in step g.

The results described above may be adulterated and, for example, comprise false-positives, in the case of cross-contamination with nucleic acids from sources other than the fluid sample. In particular, amplificates of former experiments may contribute to such undesired effects. One particular method for minimizing the effects of cross-contamination of nucleic acid amplification is described in U.S. Patent No. 5,035,996. The method involves the introduction of unconventional nucleotide bases, such as dUTP, into the amplified product and exposing carryover products to enzymatic and/or physicochemical treatment to render the product DNA incapable of serving as a template for subsequent amplifications. Enzymes for such treatments are known in the art. For example, uracil-DNA glycosylase, also known as uracil-N- glycosylase or UNG, will remove uracil residues from PCR products containing that base. The enzyme treatment results in degradation of the contaminating carryover PCR product and serves to "sterilize" the amplification reaction.

Thus, an aspect of the disclosure is the process described above, further comprising between step d) and step e) the steps of • treating the fluid sample with an enzyme under conditions in which products from amplifications of cross-contaminating nucleic acids from other samples are enzymatically degraded;

• inactivating said enzyme.

For example, the enzyme is uracil-N-glycosylase.

In a process as described above, all steps may be automated. "Automated" means that the steps of a process are suitable to be carried out with an apparatus or machine capable of operating with little or no external control or influence by a human being. Only the preparation steps for the method may have to be done by hand, e.g. storage containers have to be filled and put into place, the choice of samples has to be performed by a human being and further steps known to the expert in the field, e.g. the operation of a controlling computer. The apparatus or machine may e.g. automatically add liquids, mix the samples or carry out incubation steps at specific temperatures. Typically, such a machine or apparatus is a robot controlled by a computer which carries out a program in which the single steps and commands are specified.

A further aspect of the disclosure is an analytical system (440) for isolating and simultaneously amplifying at least two target nucleic acids that may be present in a fluid sample, said analytical system comprising the following modules:

• a separation station (230) comprising a solid support material, said separation station being constructed and arranged to separate and purify a target nucleic acid comprised in a fluid sample

• an amplification station (405) comprising at least two reaction vessels, said reaction vessels comprising amplification reagents, at least a first purified target nucleic acid in at least a first reaction vessel and at least a second purified target nucleic acid in at least a second reaction vessel, wherein the second target nucleic acid is absent from the first reaction vessel, an internal control nucleic acid and a polymerase with reverse transcriptase activity, said polymerase further comprising a mutation conferring an improved nucleic acid extension rate and/or an improved reverse transcriptase activity relative to the respective wildtype polymerase.

An "analytical system" is an arrangement of components such as instruments interacting with each other with the ultimate aim to analyze a given sample. The advantages of said analytical system are the same as described supra with respect to the process. Herein, the analytical system (440, Fig. 11) may be a system (440) comprising a module (401) for isolating and/or purifying an analyte. Further, the system (440) may additionally comprise a module (403) for analyzing said analyte to obtain a detectable signal. The detectable signal can be detected in the same module (401, 402, 403) or, alternatively, in a separate module. The term "module" as used herein relates to any spatially defined location within the analyzer (400). Two modules (401 , 403) can be separated by walls, or can be in open relationship. Any one module (401, 402, 403) can be either autonomously controlled, or control of the module (401, 402, 403) can be shared with other modules. For example, all modules are controlled centrally. Transfer between modules (401, 402, 403) can be manual, or automated. Thus, a number of different embodiments of automated analyzers (400) are disclosed.

The "separation station" is described supra.

An "amplification station" comprises a temperature-controlled incubator for incubating the contents of at least two reaction vessels. It further comprises a variety of reaction vessels like tubes or plates, in which a reaction for the analysis of the sample such as PCR takes place. The outer limits or walls of such vessels are chemically inert such that they do not interfere with the amplification reaction taking place within. For the ease of handling and to facilitate automation, the at least two reaction vessels can be combined in an integral arrangement, so they can be manipulated together.

Consequently, an aspect of the disclosure is the analytical system described above, wherein the at least two reaction vessels are combined in an integral arrangement.

Integral arrangements can e.g. be vials or tubes reversibly or irreversibly attached to each other or arranged in a rack. For example, the integral arrangement is a multiwell plate.

For example, said multiwell plate is held in a holding station. In an embodiment, one handler transports a multiwell vessel from a holding station to an air-lock (460), and a second handler transports said multiwell plate from said air-lock to said amplification station, wherein both handlers interact with said multiwell plate by a form-locking interaction.

In an embodiment, the analytical system is fully automated.

In one embodiment, at least two reaction vessels combined in an integral arrangement are transported between stations of the system.

In a second embodiment, the purified target nucleic acid is transferred from said separation station to said amplification station. For example, a pipettor comprising pipets with attached pipet tips transfers the liquid comprising the purified nucleic acid.

In a third embodiment, the purified nucleic acid is transferred from said separation station to a reaction vessel in an integral arrangement held in a holding station. For example, said reaction vessel in an integral arrangement is then transferred from said holding station to said amplification station.

The analytical system may further comprise a pipetting unit. Said pipetting unit may comprise at least one pipet, or multiple pipets. In an embodiment, said multiple pipets are combined in one or more integral arrangements, within which the pipets can be manipulated individually. Pipets can be pipets comprising pipet tips as described supra. In another embodiment, the pipets are pipetting needles.

Alternatively, a reaction vessel or arrangement of reaction vessels used for sample preparation in the separation station and containing the fluid comprising the purified target nucleic acids may be transferred from the separation station to the amplification station.

For this purpose, the analytical system may further comprise a transfer unit, said transfer unit comprising a robotic device, said device comprising a handler.

For example, the analytical system (440) described above further comprises one or more elements selected from the group consisting of:

· a detection module (403) for detecting signals evoked by an analyte

• a sealer (410)

• a storage module (1008) for reagents and/or disposables.

• a control unit (1006) for controlling system components.

A "detection module" (403) can e.g. be an optical detection unit for detecting the result or the effect of the amplification procedure. An optical detection unit may comprise a light source, e.g. a xenon lamp, optics such as mirrors, lenses, optical filters, fiber optics for guiding and filtering the light, one or more reference channels, or a CCD camera or a different camera. A "sealer" (410) is constructed and arranged to seal any vessels used in connection with the analytical system. Such a sealer can, for example, seal tubes with appropriate caps, or multiwell plates with foil, or other suitable sealing materials.

A "storage module" (1008) stores the necessary reagents to bring about a chemical or biological reaction important for analysis of the fluid sample. It can also comprise further components, e.g. disposables such as pipet tips or vessels to be used as reaction vessels within the separation station and/or the amplification station.

For example, the analytical system may further comprise a control unit for controlling system components.

Such a "control unit" (1006) may comprise software for ensuring that the different components of said analytical system work and interact correctly and with the correct timing, e.g. moving and manipulating components such as pipets in a coordinated manner. The control unit may also comprise a processor running a real-time operating system (RTOS), which is a multi-tasking operating system intended for real-time applications. In other words the system processor is capable of managing real-time constraints, i.e. operational deadlines from event to system response regardless of system load. It controls in real time that different units within the system operate and respond correctly according to given instructions.

In an embodiment, the present disclosure relates to an analytical system (440) for processing an analyte, comprising

a. a first position comprising first receptacles (1001) in linear arrangement comprising liquid samples (1010), a processing plate (101) comprising receptacles (103) in nxm arrangement for holding a liquid sample (1011), a first pipetting device (700) comprising at least two pipetting units (702) in linear arrangement, wherein said pipetting units (702) are coupled to pipette tips (3, 4), and a tip rack (70) comprising pipette tips (3, 4) in an ax(nxm) arrangement;

b. a second position comprising a holder (201, 128) for said processing plate (101), a holder (330) for a multiwell plate, a holder (470) for said tip rack (70) and a second pipetting device

(35), said second pipetting device (35) comprising pipetting units (702) in an nxm arrangement for coupling to pipette tips (3, 4) (Fig. 12). The term "holder" as used herein relates to any arrangement capable of receiving a rack or a processing plate.

The advantages of the analytical system (440) are as described above for the methods.

For example, the position of said pipetting units (702) of the first pipetting device (700) are variable. Exemplary embodiments of said first pipetting device (700) are described hereinafter.

In one embodiment, the tip rack (70) comprises pipette tips (3, 4) in an ax(nxm) arrangement. For example, a first type (4) and a second type (3) of pipette tips are comprised in the tip rack (70). In this embodiment, the first type of pipette tips (4) is arranged in an nxm arrangement, and the second type of pipette tips (3) is arranged in the nxm arrangement. In this context, "n" denotes the number of rows and m the number of columns, wherein n is 6 and m is 8. For example, the first type of pipette tips (4) has a different volume than the second type of pipette tips (3), or, the volume of the first type of pipette tips (4) is more than 500 ul, and the volume of the second type of pipette tips (3) is less than 500 ul. In this embodiment, a=2. However, more than two types of pipette tips, and thus a >2 may be present.

In one aspect, the analytical system (440) comprises a control unit (1006) for allocating sample types and individual tests to individual positions of said processing plate (101). For example, said positions are separate cells (401, 402). In one aspect, the system additionally comprises a transfer system (480) for transferring said process plate (101) and said rack (70) between first (402) and second (401) positions. Embodiments of said transfer system (480) are conveyor belts or, one or more handler.

Furthermore, for example said pipette units of said second pipetting device (35) are engaged to pipette tips (3, 4) which were used in the first position (402).

An embodiment of the system (440) additionally comprises a third station (403) comprising a temperature-controlled incubator for incubating said analyte with reagents necessary to obtain a detectable signal. Further embodiments of this system are described hereinafter.

More optimal control of the allocation of samples and tests to the nxm arrangement is achieved with a first processor (1004) which is comprised in said first position (402) to which said control unit (1006) transfers instructions for allocating sample types and individual tests to specific positions in the nxm arrangement of vessels (103) of the process plate (101), and a second processor (1005) which is comprised in said second position (401) to which said control unit (1006) transfers instructions for allocating sample types and individual tests to specific positions in the nxm arrangement of vessels (103) of the process plate.

For example, said system additionally comprises a first processor located in said first position, and a second processor located in said second position.

For example, said first processor (1004) controls said first pipetting device (700) and said second processor (1005) controls said second pipetting device (35). DESCRIPTION OF THE FIGURES

Figure 1:

Schematic depiction of the sample preparation workflow as used in an embodiment of the invention.

Arrows pointing down denote addition of a component or reagent to each respective well of the deepwell plate mentioned above, arrows pointing up their respective removal. These actions were performed manually in steps 2, 3, 4, 21 and 22, by the process head of the apparatus in steps 10, 14, 16, 18, and 24, and by the reagent head of the apparatus in steps 5, 6, 7, 11, 15 and 19.

It has to be understood that the volumes used can be adjusted flexibly within the spirit of the invention, for example at least about up to 30% of the disclosed values. In particular, in the case of step 2, the sample volume can be variable in order to take into account the different types of fluid samples which may require more or less starting material for obtaining proper results, as known by the artisan. For example, the range is from about 100 ul to about 850 ul. Or, it is about 100 ul, about 500 ul or about 850 ul. For example, the volume in the respective vessels is adjusted to an identical total volume with the diluent in step 3. For example, as in the scheme shown in Fig. 1, the total volume adds up to about 850 ul.

Figure 2:

Growth curves of the amplifications of the target nucleic acids derived from HIV, HBV and CT carried out on a LightCycler480 (Roche Diagnostics GmbH, Mannheim, DE) as described in Example 1. The "Signal" indicated on the y-axis is a normalized fluorescent signal. The x- axis shows the number of the respective PCR cycle.

The growth curves of HIV and HBV are shown along with the growth curves of the corresponding internal control nucleic acid. The respective target nucleic acid curves are represented by straight lines, the control nucleic acid curves by dotted lines.

Fig. 2a: Qualitative HIV assay, measured in the channel for detection of the target probe.

Fig. 2b: Qualitative HIV assay, measured in the channel for detection of the control probe.

Fig. 2c: Quantitative HIV assay, measured in the channel for detection of the target probe. Fig. 2d: Quantitative HIV assay, measured in the channel for detection of the control probe.

Fig. 2e: Quantitative HBV assay, measured in the channel for detection of the target probe.

Fig. 2f: Quantitative HBV assay, measured in the channel for detection of the control probe.

Fig. 2g: CT assay, measured in the channel for detection of the target probe.

Figure 3: Perspective view of the processing plate. Figure 4: Perspective view of the processing plate from the opposite angle.

Figure 5: Top view of the processing plate.

Figure 6: Cross-sectional view along the longer side of the processing plate.

Figure 7: A partial view of the cross-sectional view.

Figure 8: Perspective view of the longer side of the processing plate. Figure 9: a) to d) show different views of the second embodiment of the magnetic separation station.

Figure 10: (a) to (c) show a view of the first embodiment of the magnetic separation station holding the Processing plate, with the first type of magnets in the uppermost Z- position, and the second type of magnets in the lowermost Z-position. Figure 11: Schematic drawings of an analyzer comprising different stations, modules or cells. Figure 12: Shows an analytical system of the present disclosure.

Figure 13: Linearity of the quantitative HBV assay in EDTA plasma according to the data in

Example 2.

Figure 14: Linearity of the quantitative HBV assay in serum according to the data in

Example 2.

Figure 15: Linearity of the quantitative HCV assay in EDTA plasma according to the data in

Example 2.

Figure 16: Linearity of the quantitative HCV assay in serum according to the data in

Example 2.

Figure 17: Linearity of the quantitative HIV assay in EDTA plasma according to the data in

Example 2.

EXAMPLES

The following examples are offered to illustrate, but not to limit the claimed invention. Example 1:

This example describes a process for isolating and simultaneously amplifying at least a first and a second target nucleic acid using a single generic internal control nucleic acid.

In brief, in the depicted embodiment, realtime PCR is carried out simultaneously and under identical conditions on a panel of several different targets comprising bacteria (Chlamydia trachomatis, CT) as well as a DNA virus (HBV) and an RNA virus (HIV). All samples were processed and analyzed within the same experiment, i.e. on the same deepwell plate (for sample preparation) or multiwell plate (for amplification and detection), respectively.

The following samples were prepared and subsequently analyzed:

Suitable standards or other types of targets are available to the skilled artisan.

The instruments listed in the following table were used according to the instructions of the respective manufacturer: Instrument Manufacturer

Hamilton Star Hamilton Medical AG (Bonaduz, CH)

Light Cycler 480 Roche Diagnostics GmbH (Mannheim, DE)

Chameleon Sealer K biosystems (Essex, UK)

Compressor K biosystems (Essex, UK)

For sample preparation the following reagents were used as diluents:

The following dilutions were prepared in advance and stored overnight (plasma dilutions at - 60 to -90°C, PreservCyt dilutions at 2-8°C):

Each respective sample (500 ul) and each respective specimen diluent (350 ul) were pipetted manually into a deepwell plate, wherein each sample was added to three different wells for triplicate analysis. To each well containing an HIV or HBV sample, 50 ul of an internal control nucleic acid were manually added. For the qualitative HIV assay, an RNA serving as a qualitative control was added (100 armored particles/sample). For the quantitative HIV assay, an RNA serving as a quantitative standard was added (500 armored particles/sample). For the quantitative HBV assay, a DNA serving as a quantitative standard was added (1E4 copies/sample). The sequence of said control nucleic acids was identical in all cases and selected from the group of SEQ ID NOs 45-48.

The respective control nucleic acid was stored in the following buffer:

IC/IQS - Storage Buffer Cone, or pH

Tris (mM) 10

EDTA (mM) 0.1

Sodium Azide (w/v, %) 0.05

Poly rA RNA (mg/1) 20

pH 8 Sample preparation was performed on a Hamilton Star (Hamilton, Bonaduz, CH), following the workflow according to the scheme depicted in Fig. 1 and using the following reagents:

Protease reagent Cone, or pH

Tris (mM) 10

EDTA (mM) 1

Calcium Chloride (mM) 5

Calcium Acetate (mM) 5

Esperase (mg/ml) 80

Glycerin (w/v, %) 50

pH 5.5

MGP Reagent Cone, or pH

MPG Powder (mg/ml) 60

Tris (mM) 30

Methylparaben (w/v, %) 0.1

Sodium Azide (w/v, %) 0.095

pH 8.5

Lysis Reagent Cone, or pH

Guanidine Thiocyanate (M) 4

Sodium Citrate (mM) 50

Polydocanol (w/v, %) 5

Dithiotreitol (w/v, %) 2

pH 5.8

Wash buffer Cone, or pH

Sodium Citrate (mM) 7.5

Methylparaben (w/v, %) 0.1

pH 4.1 Elution buffer Cone, or pH

Tris (niM) 30

Methylparaben (w/v, %) 0.2

pH 8.5

After the final step, the process head of the Hamilton Star apparatus added the respective mastermixes (Mmxs) containing amplification reagents to each well, mixed the fluids containing the isolated nucleic acids with the Mmx and transferred each resulting mixture to a corresponding well of a microwell plate in which the amplification was carried out.

The following mastermixes (each consisting of the two reagents Rl and R2) were used:

For HIV:

Concentration / 50 μΐ-

Rl Reagent

PCR [μΜ]

Water (PCR grade)

Mn(Ac) ₂ * 4H ₂ 0 (pH 6.1 adjusted with Acetic Acid) 3Ό00

NaN3/Ri, buffered with 10 mM Tris at pH7 [%] 0.018

Concentration / 50 μΐ-

R2 Reagent

PCR [μΜ]

DMSO [%] 5.000 %

NaN3/Ri, buffered with 10 mM Tris at pH7 [%] 0.027 %

Potassium acetate pH 7.0 Π0Ό00

Glycerol [%] 3.000 %

Tricine pH 8.0 50Ό00

Igepal [%] 0.024 %

dGTP 337.5

dATP 337.5

dCTP 337.5

dUTP 675

Primers/probes selected from SEQ ID NOs 1-35 0.1-0.15

SEQ ID NO 42 0.1

SEQ ID NO 43 0.1 SEQ ID NO 44 0.1

Uracil-N-Glycosylase 10 (U/reaction)

Z05-D Polymerase 40 (U/reactionn)

NTQ21-46A - Aptamer 0.222

Water or HBV:

R2 Reagent Concentration / 50 μΙ-PCR

H ₂ 0 100 %

Tricine 7.7 40 mM

Tween 0.03 % (v/v)

Glycerol 5 % (v/v)

KOH 25.2 mM

KOAc 121.8 mM

NTQ21-46A (Aptamer) 0.2625 uM dGTP 0.42 uM dATP 0.42 uM dCTP 0.42 uM dUTP 0.84 uM

SEQ ID NO 36 1.2 uM

SEQ ID NO 37 0.1 uM

SEQ ID NO 38 1.2 uM

SEQ ID NO 42 0.6 uM

SEQ ID NO 43 0.6 uM

SEQ ID NO 44 0.15 uM

Z05D Polymerase 35 (U/reaction)

Uracil-N-Glycosylase 2 (U/reaction)

Sodium Azide 0.027 % (m/v)

Rl Reagent Concentration / 50 μΙ-PCR

H ₂ 0 100 %

MgOAc 2.5 mM

MnOAc pH6.1 2.5 mM

Sodium Azide 0.018 % (m/v) For CT:

Rl Reagent Concentration / 50 μΙ-PCR

Water (PCR grade)

Mn(Ac) ₂ (pH 6.5 in 0.002% (V/V) Glacial Acetic Acid) 2.7 mM

NaN ₃ 0.0135% (W/V)

R2 Reagent Concentration / 50 μΙ-PCR

NaN ₃ /Ri, buffered with 10 mM Tris at pH7 [%] 0.0315%

Potassium acetate 112.4 mM

Glycerol [%] 3.5%

Tricine 61 mM

Potassium hydroxide 28.4 mM

dGTP 525 uM

dATP 525 uM

dCTP 525 uM

dUTP 1.05 mM

SEQ ID NO 39 750 nM

SEQ ID NO 40 600 nM

SEQ ID NO 41 116 nM

Aptamer NTQ-46A 175 nM

Uracil-N-Glycosylase 5 U/reaction

Z05-D Polymerase 31 U/reaction

For amplification and detection, the microwell plate was sealed with an automated plate sealer (see above), and the plate was transferred to a LightCycler 480 (see above).

The following PCR profile was used:

Thermo cycling profile

Program Target Acquisition Hold Ramp Rate Analysis

Name (°C) Mode (hh:mm:ss) (°C / s) Cycles Mode

Pre-PCR 50 None 00:02:00 4.4

94 None 00:00:05 4.4

55 None 00:02:00 2.2 1 None

60 None 00:06:00 4.4

65 None 00:04:00 4.4 1st

Quantifi¬

Measure95 None 00:00:05 4.4 5

cation ment 55 Single 00:00:30 2.2

2nd

Quantifi¬

Measure91 None 00:00:05 4.4 45

cation ment 58 Single 00:00:25 2.2

Cooling 40 None 00:02:00 2.2 1 None

Detection Format (Manual)

Filter Combination Integration Time (sec)

435 ^■ - 470 1

495 ^■ - 525 0.5

540 - - 580 0.5

610 - - 645 0.5

680 - - 700 1

The Pre-PCR program comprises initial denaturing and incubation at 55, 60 and 65°C for reverse transcription of RNA templates. Incubating at three temperatures combines the advantageous effects that at lower temperatures slightly mismatched target sequences (such as genetic variants of an organism) are also transcribed, while at higher temperatures the formation of RNA secondary structures is suppressed, thus leading to a more efficient transcription.

PCR cycling is divided into two measurements, wherein both measurements apply a one-step setup (combining annealing and extension). The first 5 cycles at 55°C allow for an increased inclusivity by pre-amplifying slightly mismatched target sequences, whereas the 45 cycles of the second measurement provide for an increased specificity by using an annealing/extension temperature of 58°C.

Using this profile on all samples comprised on the microwell plate mentioned above, amplification and detection was achieved in all samples, as depicted in Fig. 2. This shows that the sample preparation prior to amplification was also successfully carried out.

The results for the qualitative and quantitative HIV internal controls and the quantitative HBV internal control are depicted separately in Fig. 2 for the sake of clarity. It can be seen that the controls were also successfully amplified in all cases. The quantitation of the HIV and HBV targets in the quantitative setup were calculated by comparison with the internal control nucleic acid serving as a quantitative standard.

Example 2:

The generic amplification process described hereinabove was carried out on a variety of different target nucleic acids in separate experiments but under identical conditions. Isolation of the respective nucleic acid was carried out as described under Example 1.

The respective generic internal control nucleic acid was selected from SEQ ID NOs 45-49 and was armored RNA for RNA targets and lambda-packaged DNA for DNA targets. For qualitative RNA assays, 300 particles were added per sample, for quantitative RNA assays 3000 and for all DNA assays 500.

The following PCR profile was used on all targets:

In detail, the following experiments were performed: 1. Qualitative multiplex analysis of HBV, HCV and HIV

a. Mastermix

Rl:

Analytical Sensitivity / LOD

For each detected virus (HIV-1 group M, HIV-1 group O, HIV-2, HBV and HCV), at several concentrations/levels at and around the anticipated LOD for EDTA-plasma. One panel per virus and concentration was tested with at least 20 valid replicates per concentration. The LOD was determined by PROBIT analysis (see Table 1-5). HIV

Table 1: HIV-1 Group M Hit rates and Probit LOD from individual panel

Titer of WHO Standard for HIV-1 Group M was converted to IU/mL.

Therefore HIV-1 Group M LOD in IU/mL is

LOD by PROBIT analysis (95% hitrate): 6.77 IU/mL

95% confidence interval for LOD by PROBIT analysis: 4.75 - 15.4 IU/mL Table 2: HIV-1 Group O Hit rates and Probit LOD from individual panel

Concentration Number of replicates Number of positives Hit rate

60 cp/mL 21 21 100 %

30 cp/mL 20 20 100 %

20 cp/mL 21 21 100 %

14 cp/mL 21 19 90 %

7 cp/mL 21 15 71 %

4.5 cp/mL 21 12 57 %

0 cp/mL (neg. control) 12 0 0 % LOD by PROBIT analysis (95% hitrate) 14.9 cp/mL

95% confidence interval for LOD by PROBIT analysis 10.9 - 31.5 cp/mL

Titer of Primary Standard for HIV-1 Group O was reassigned to CBER HIV-1 Group O panel; calculation factor is 0.586.

Therefore HIV-1 Group O LOD is

LOD by PROBIT analysis (95 % hitrate) : 8.8 cp/mL

95% confidence interval for LOD by PROBIT analysis: 6.4 - 18.5 cp/mL

Table 3: HIV-2 Hit rates and Probit LOD from individual panel

Titer of Primary Standard for HIV-2 was reassigned to CBER HIV-2 panel; calculation factor is 26.7.

Therefore HIV-2 LOD is

LOD by PROBIT analysis (95% hitrate): 34.44 cp/mL

95% confidence interval for LOD by PROBIT analysis: 21.89 - 83.04 cp/mL

HBV

Table 4: HBV Hit rates and Probit LOD from individual panel

Concentration Number of replicates Number of positives Hit rate

7.6 IU/mL 21 21 100 %

3.8 IU/mL 21 21 100 %

1.9 IU/mL 21 20 95 % 0.95 IU/mL 21 14 67 %

0.6 IU/mL 19 12 63 %

0.4 IU/mL 21 12 57 %

0 IU/mL (neg. control) 12 0 0 %

LOD by PROBIT analysis (95% hitrate) 2.27 IU/mL

95% confidence interval for LOD by PROBIT analysis 1.48 - 6.54 IU/mL

HCV

Table 5: HCV Hit rates and Probit LOD from individual panel

2. Qualitative analysis of WNV

Mastermix

Rl:

R2:

Reagent Cone, in 50 ul-PCR (uM)

DMSO (%) 5.4

NaN3/Ri, buffered with 10 mM Tris at pH7 0.027 K acetate H 7.0 120Ό00

Glycerol (%) 3

Tween 20 (%) 0.015

Tricine pH 8.0 60Ό00

NTQ21-46A - Aptamer 0.2222

Uracil-N-Glycosylase (U/uL) 0.2

dGTP 400.0

dATP 400.0

dCTP 400.0

dUTP 800.0

Z05-D Polymerase (U/ul)* 0.9

Primers/probes selected from SEQ ID NOs 53-59 0.08-0.4

SEQ ID NO 42 0.150

SEQ ID NO 43 0.150

SEQ ID NO 44 0.100

Analytical Sensitivity / LOD

For the viruses (WNV, SLEV and JEV) an independent panel was prepared as a dilution series of the respective Standard including several concentrations/levels at and around the anticipated LOD. One panel per virus and concentration was tested with at least 20 valid replicates per concentration. The LOD was determined by PROBIT analysis.

Table 6: WNV Hit rates and Probit LOD from individual panel

Concentration Number of replicates Number of positives Hit rate

20 cp/mL 21 21 100 %

12 cp/mL 21 21 100 %

8 cp/mL 21 21 100 %

5 cp/mL 21 17 81 %

2.5 cp/mL 21 15 71.4 %

0.5 cp/mL 21 1 4.8 %

0 cp/mL (neg. control) 12 0 0 %

LOD by PROBIT analysis (95% hitrate) 6.57 cp/mL

95% confidence interval for LOD by PROBIT analysis 4.74 - 11.03 cp/mL Table 7: SLEV Hit rates and Probit LOD from individual panel

Table 8: JEV Hit rates and Probit LOD from individual panel

3. Quantitative analysis of HBV

Mastermix Rl:

Reagent Final Cone, in 50 ul-PCR (uM)

Mn(Ac)2 * 4H20 (pH 6.1 adjusted with Acetic Acid) 3 ^* 300

NaN3/Ri, buffered with 10 mM Tris at pH7 0.018

pH 6.41 R2:

Reagent Final Cone, in 50 ul-PCR (uM)

Glycerol (%, w/v) 3%

Tricine 60 mM

DMSO (%, v/v) 5.4%

KOAc 120 mM

Tween 20 (v/v) 0.015%

Aptamer NTQ21-46 A 0.222 μΜ

Z05D Polymerase 0.9 υ/μΐ, (45 U/rxn)

Uracil-N-Glycosylase 0.2 U^L (10 U/rxn)

Sodium Azide (w/v) 0.027%

dCTPs 400 μΜ

dGTPs 400 μΜ

dATPs 400 μΜ

dUTPs 800 μΜ

SEQ ID NO 36 1.2 μΜ

SEQ ID NO 37 1.2 μΜ

SEQ ID NO 50 0.6 μΜ

SEQ ID NO 51 0.6 μΜ

SEQ ID NO 38 0.1 μΜ

SEQ ID NO 52 Μ

Analytical Sensitivity / LOD

Four dilution panels were prepared with HBV Secondary Standard (representing Genotype A), i.e., two in HBV negative serum for sample input volumes of 200 μΐ, and 500 μί, and two in HBV negative EDTA-plasma for sample input volumes of 200 μΐ _^ and 500 μΐ _^ . Each panel included 7 concentration levels at and around the anticipated LOD. One panel per matrix was tested with > 21 replicates per concentration level. At least 20 replicates needed to be valid. The LOD was determined by PROBIT analysis at 95% hit rate and by > 95% hit rate analysis. Table 9: LOD analysis for 200 μΐ _^ input volume in EDTA-plasma. *

Concentration Number of replicates Number of positives Hit rate

25 IU/mL 41 41 100 %

15 IU/mL 41 39 95.1 %

10 IU/mL 41 40 97.6 %

7 IU/mL 41 40 97.6 %

4 IU/mL 24 20 83.3 % 1 IU/mL 24 4 16.7 %

0 IU/mL (neg. control) 24 0 0 %

LOD by PROBIT analysis (95% hitrate) 8.2 IU/m]

95% confidence interval for LOD by PROBIT analysis 4.8 - 26.0 IU/mL

* Additional replicates were tested to narrow the observed 95% confidence interval.

Table 10: LOD analysis for 500μί input volume in EDTA-plasma.

Table 11: LOD analysis for 200μί input volume in serum.

Concentration Number of replicates Number of positives Hit rate

25 IU/mL 21 21 100 %

15 IU/mL 21 20 95.2 %

10 IU/mL 21 21 100 %

7 IU/mL 21 20 95.2 %

4 IU/mL 21 15 71.4 %

1 IU/mL 21 8 38.1 %

0 IU/mL (neg. control) 21 0 0 %

LOD by PROBIT analysis (95% hitrate) 9.4 IU/m]

95% confidence interval for LOD by PROBIT analysis 6.2 - 19.0 IU/mL Table 12: LOD analysis for 500μί input volume in serum.

Summary LOD:

EDTA-plasma: The PROBIT analysis at 95 % hit rate resulted in an LOD of 8.2 IU/mL for 200 sample input volume and 2.3 IU/mL for 500 sample input volume for EDTA- plasma.

The 95 % confidence interval range for these concentrations was 4.8 - 26.0 IU/mL for 200 μΐ _^ sample input volume and 1.6 - 4.2 IU/mL for 500 μΐ _^ sample input volume.

Serum: The PROBIT analysis at 95 % hit rate resulted in an LOD of 9.02 IU/mL for 200 μΐ, sample input volume and 4.1 IU/mL for 500 μΐ _^ sample input volume for serum.

The 95 % confidence interval range for these concentrations was 6.2 - 19.0 IU/mL for 200 μΐ _^ sample input volume and 2.4 - 10.0 IU/mL for 500 μΐ _^ sample input volume.

Linearity

One EDTA-plasma panel and one serum panel were prepared by using HBV genotype A (provided by RMD Research Pleasanton, linearized plasmid, pHBV-PC_ADW2). Each of the panels was analyzed at 12 concentration levels for the determination of the expected dynamic range (4 - 2E+09 IU/mL) of the assay. All concentration levels/panel members (PM) were tested in 21 replicates.

This study was done with a sample input volume of 500 μΐ,. The concentration levels were selected as follows: One level below expected Lower Limit of Quantitation (LLOQ), one at expected LLOQ, one above expected LLOQ, several concentrations at intermediates levels, at expected Upper Limit of Quantitation (ULOQ) and one above expected ULOQ.: PM 12 - 2.0E+09 IU/mL - above expected ULOQ

PM 11 - 1.OE+09 IU /niL - at expected ULOQ

PM 10 - 1.0E+08 IU /niL - below expected ULOQ

PM 9 - 1.0E+07 IU /mL - intermediate concentration level

PM 8 - 1.0E+06 IU /mL - intermediate concentration level

PM 7 - 1.0E+05 IU /mL - intermediate concentration level

PM 6 - 1.0E+04 IU /mL - intermediate concentration level

PM 5 - 1.0E+03 IU /mL - intermediate concentration level

PM 6a - 2.0E+02 IU/mL - intermediate concentration level (PM 6 diluted to 2.0E+02 IU/mL, used for titer assignment of serum panel)

PM 4 - 1.0E+02 IU /mL - intermediate concentration level (also used for titer assignment of plasma panel)

PM 3 - 5.0E+01 IU/mL - above expected LLOQ

PM 2 - l .OE+01 IU /mL - at expected LLOQ

PM 1 - 4.0E+00 IU /mL below expected LLOQ

For every valid sample of the linearity panel, the observed HBV DNA titer was transformed to log 10 titer and the mean log 10 titer was calculated per concentration level.

Table 13: Linearity in EDTA Plasma

Nominal Titer Assigned Titer Assigned LoglO Mean Log 10 Replicates (IU/mL) (IU/mL) Titer Titer observed

4.00E+00 3.50E+00 0.54 0.52 17 l .OOE+01 8.70E+00 0.94 0.91 21

5.00E+01 4.40E+01 1.64 1.69 21

1.00E+02 8.70E+01 1.94 2.04 21

1.00E+03 8.70E+02 2.94 3.01 21

1.00E+04 8.70E+03 3.94 3.9 21

1.00E+05 8.70E+04 4.94 4.88 21

1.00E+06 8.70E+05 5.94 5.87 21

1.00E+07 8.70E+06 6.94 6.92 21

1.00E+08 8.70E+07 7.94 8.01 21

1.00E+09 8.70E+08 8.94 9.04 21

2.00E+09 1.70E+09 9.24 9.38 21 A graphical depiction of this result is shown in Fig. 13.

Table 14: Linearity in Serum

A graphical depiction of this result is shown in Fig. 14.

Summary Linearity:

The linear range, defined as the concentration range for which the log 10 deviation of the mean loglO observed titers is within ± 0.3 of the loglO nominal titer was determined as: 3.5E+00 IU/mL - 1.7E+09 IU/mL for EDTA-plasma and 3.3E+00 IU/mL - 1.7E+09 IU/mL for serum. The Lower Limit of Quantitation was found to be: 4.0E+00 IU/mL for EDTA- plasma and serum.

4. Quantitative analysis of HCV

Mastermix Rl:

Reagent Final Cone, in 50 ul-PCR (uM)

Mn(Ac)2 * 4H20 (pH 6.1 adjusted with Acetic Acid) 3 ^* 300

NaN3/Ri, buffered with 10 mM Tris at pH7 0.018

pH 6.41 R2:

Reagent Final Cone, in 50 ul-PCR

Glycerol (%, w/v) 3%

Tricine 60 mM

DMSO (%, v/v) 5.4%

KOAc 120 mM

Tween 20 (v/v) 0.015%

NTQ21-46 A 0.222 μΜ

Z05D 0.9 \]/μΙ (45 U/rxn)

UNG 0.2 U^L (10 U/rxn)

Sodium Azide (w/v) 0.027

dCTPs 400 μΜ

dGTPs 400 μΜ

dATPs 400 μΜ

dUTPs 800 μΜ

Primers/probes selected from SEQ ID NOs 60-76 0.1 μΜ

SEQ ID NO 42 0.3 μΜ

SEQ ID NO 43 0.3 μΜ

SEQ ID NO 44 μΜ

Analytical Sensitivity / LOD

A dilution panel was prepared with Roche HCV Secondary Standard in HCV negative EDTA plasma and serum using a sample input volumes of 200 μΐ _^ and 500 μΐ _^ . Each concentration level was tested with 21 replicates. At least > 20 replicates have to be valid. The LOD was determined by PROBIT analysis at 95% hit rate and by > 95% hit rate analysis.

Table 15: Hit rates and Probit with 200 μΐ _^ sample process input volume for EDTA-plasma

Concentration Number of replicates Number of positives Hit rate

55 IU/mL 21 21 100 %

38 IU/mL 21 21 100%

25 IU/mL 21 20 95 %

12.5 IU/mL 21 19 90 %

6 IU/mL 21 15 71 %

3 IU/mL 21 6 29 %

0 IU/mL (neg. control) 21 0 0 %

LOD by PROBIT analysis (95% hitrate) 17.4 IU/mL

95% confidence interval for LOD by PROBIT analysis 12.1 - 34.3 IU/mL Table 16: Hit rates and Probit with 500 μΐ _^ sample process input volume for EDTA-plasma

Table 17: Hit rates and Probit with 200 sample process input volume for serum

Concentration Number of replicates Number of positives Hit rate

55 IU/mL 21 21 100 %

38 IU/mL 21 21 100%

25 IU/mL 21 20 95 %

12.5 IU/mL 21 18 86 %

6 IU/mL 21 13 62 %

3 IU/mL 21 6 29 %

0 IU/mL (neg. control) 21 0 0 %

LOD by PROBIT analysis (95% hitrate) 20.2 IU/m]

95% confidence interval for LOD by PROBIT analysis 14.0 - 39.3 IU/mL

Table 18: Hit rates and Probit with 500 sample process input volume for serum

Concentration Number of replicates Number of positives Hit rate

22 IU/mL 21 21 100 %

15 IU/mL 21 21 100%

10 IU/mL 21 20 95 %

5 IU/mL 21 18 86 %

2.5 IU/mL 21 12 57 % 1 IU/mL 21 4 19 %

0 IU/mL (neg. control) 21 0 0 %

LOD by PROBIT analysis (95% hitrate) 8.2 IU/mL

95% confidence interval for LOD by PROBIT analysis 5.8 - 15.0 IU/mL

Summary LOD:

1. The PROBIT analysis at 95 % Hit rate resulted in an LOD of 17.4 IU/mL for 200 sample process input volume and 9.0 IU/mL for 500 sample process input volume for EDTA plasma. The 95 % confidence interval for these concentrations is 12.1 - 34.3

IU/mL for 200 sample process input volume and 5.5 - 25.4 IU/mL for 500 μΐ, sample process input volume.

2. The values of the PROBIT analysis at 95 % Hit rate is 20.2 IU/mL for 200 μΐ _^ sample process input volume and 8.2 IU/mL for 500 μΐ, sample process input volume for serum. The 95 % confidence interval for these concentrations is 14.0 - 39.3 IU/mL for 200 μΐ _^ sample process input volume and 5.8 - 15.0 IU/mL for 500 μΐ, sample process input volume.

Linearity

One preparation of an EDTA-plasma panel and one preparation of a serum panel of HCV aRNA traceable to the HCV WHO Standard were analyzed. The linearity panels were prepared by serial dilution and analyzed at 10 different concentrations. The study was done with 500 μΐ, sample process input volume. The concentrations were selected as follows: One level below expected Lower Limit of Quantification (LLoQ), one at LLoQ, one above LLOQ, several concentrations at intermediates levels, at expected Upper Limit of Quantification (ULoQ) and one at or above ULoQ. For all concentrations 21 replicates were tested.

PM 1 - 2.0E+08 IU/mL - above expected ULoQ

PM 2 - 1.0E+08 IU /mL - at expected ULoQ

PM 3 - 1.0E+07 IU /mL - below expected ULoQ

PM 4 - 1.OE+06 IU /mL - intermediate concentration level

PM 5 1.OE+05 IU /mL - intermediate concentration level

PM 6 - 1.0E+04 IU /mL - intermediate concentration level for titer assignment PM 7 - 1.0E+03 IU /mL - intermediate concentration level PM 8 - 1.0E+02 IU /mL - above expected LLoQ

PM 9 - l .OE+01 IU /mL - at expected LLoQ

PM 10 - 8.0E+00 IU /mL - below expected LLoQ

Table 19: Linearity in EDTA Plasma

A graphical depiction of this result is shown in Fig. 15.

Table 20: Linearity in Serum

A graphical depiction of this result is shown in Fig. 16. Summary Linearity:

The linear range, defined as the concentration range for which the log 10 deviation of the mean loglO observed titers is within ± 0.3 of the loglO nominal titer was determined as: 4.87E+00 IU/mL - 1.22E+08 IU/mL for EDTA-plasma and 3.90E+00 IU/mL - 9.92E+07 IU/mL for serum.

5. Quantitative analysis of HIV

Mastermix

Rl:

Analytical Sensitivity / LOD

A dilution panel was prepared with HIV-IM Secondary Standard in HIV-1 negative EDTA plasma for sample input volumes of 200 μΐ _^ and 500 μΐ _^ . Each concentration level was tested with 21 replicates. At least > 20 replicates have to be valid. The LOD was determined by PROBIT analysis at 95% hit rate and by > 95% hit rate analysis.

Table 21: LOD analysis for 200 input volume in EDTA-plasma

Table 22: LOD analysis for 500 input volume

Summary LOD

1. The PROBIT analysis at 95 % hit rate resulted in an LOD of 41.8 cp/mL for 200 input volume and 18.9 cp/mL for 500 μΐ, input volume. 2. The 95% confidence interval range for these concentrations was 30.9 - 74.9 cp/mL for 200 μΐ, input volume and 14.9 - 29.4 cp/mL for 500 μΐ, input volume.

Linearity

The samples used in the Linearity / Dynamic Range / Accuracy study consisted of a dilution panel of an HIV-1 cell culture supernatant material, HIV-1 group M subtype B.

The linearity panel was prepared by serial dilution. This panel was analyzed at 10 concentration levels.

The concentrations were selected as follows: One level below expected Lower Limit of Quantitation (LLoQ), one at LLoQ, one above LLoQ, several concentrations at intermediate levels, at expected Upper Limit of Quantitation (ULoQ) and one above ULoQ. For all concentrations 21 replicates were tested. The linearity study was done with 500 input volume):

PM 1 - 2.0E+07 cp/mL - above expected ULoQ

PM 2 - 1.0E+07 cp/mL - at expected ULoQ

PM 3 - 1.OE+06 cp/mL - below expected ULoQ

PM 4 - 1.0E+05 cp/mL - intermediate concentration level

PM 5 3.0E+04 cp/mL - intermediate concentration level for titer assignment

PM 6 - 1.0E+04 cp/mL - intermediate concentration level

PM 7 - 1.0E+03 cp/mL - intermediate concentration level

PM 8 - 1.0E+02 cp/mL - intermediate concentration level

PM 9 - 5.0E+01 cp/mL - above expected LLoQ

PM 10 - 2.0E+01 cp/mL - at expected LLoQ

PM 11 - 1.5E+01 cp/mL below expected LLoQ Table 23: Linearity in EDTA Plasma

Nominal Titer Assigned Titer Assigned LoglO Mean Log 10 Replicates (cp/mL) (cp/mL) Titer Titer observed

1.50E+01 1.50E+01 1.2 1.3 21

2.00E+01 2.00E+01 1.3 1.5 21

5.00E+01 5.10E+01 1.7 1.8 21

1.00E+02 1.00E+02 2 2 21

1.00E+03 1.00E+03 3 3 21 1.00E+04 1.00E+04 4 4 21

1.00E+05 1.00E+05 5 5 21

1.00E+06 1.00E+06 6 6 21

1.00E+07 1.00E+07 7 7 21

2.00E+07 2.00E+07 7.3 7.4 21

A graphical depiction of this result is shown in Fig. 17.

Summary Linearity

The linear range, defined as the concentration range for which the log 10 deviation of the mean log 10 observed titers is within ± 0.3 of the log 10 nominal titer was determined as 1.5E+01 cp/mL - 2.0E+07 cp/mL.

Example 3:

Using the generic amplification process described hereinabove for the amplification of nucleic acids (both RNA and DNA) can lead to overquantification if the desired target nucleic acid is DNA but there is a significant amount of RNA present such that amplification of the undesired RNA can occur. One method to eliminate the undesired amplification of RNA is to employ RNase H which degrades RNA that is present in a RNA-DNA hybrid complex. An experiment was therefore performed for the quantitative analysis of HBV as described in Example 2, Section 3 with the exception of the addition in the R2 Mastermix reagent of 1 unit (per 50 μΐ PCR reaction) of either Hybridase™ Thermostable RNase H (Epicentre, Madison, WI) or recombinant E.Coli RNase H (New England Biolabs, Ipswich, MA). Amplification was performed on an RNA template (between 30,000 and 3,000,000 copies per reaction) or on an DNA template (between 30 and 3000 copies per reaction). The results of the experiment are shown on Table 24. Reactions with the DNA template in the presence of either RNase H enzyme showed no difference in amplification efficiency compared to reactions in the absence of RNase H. In contrast, the amplification of the RNA template was severely delayed or even absent (for Hybridase™), clearly showing that the RNA template was degraded by the addition of the RNase H enzyme prior to amplification. Table 24: Effect of RNase H on RNA and DNA amplification

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art.