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Title:
NEW NUCLEOTIDE SEQUENCE AMPLIFICATION METHOD
Document Type and Number:
WIPO Patent Application WO/2011/131192
Kind Code:
A1
Abstract:
Current method of choice for virus identification in many diagnostic laboratories is specific real-time polymerase chain reaction (PCR), where sequence-specific primer pairs are used for each virus, or a group of viruses. This is a rapid, sensitive and specific method that is easy to perform, but can have limited sensitivity that may lead to falsely negative results. A random pre-amplification of the sample prior to a specific PCR could be helpful in clinical cases where the detection limit of the real-time PCR assay is not sensitive enough. This could be when only very limited amount of sample is available or where the pathogen is present at very low amounts. The present invention discloses a nucleotide amplification method comprising of random unbiased whole genome amplification (WGA) pre-amplification followed by a specific amplification performed in the same vial or tube. The two reaction mixtures are separated by a wax layer so the reaction mixture on top of the wax layer is the pre-amplification mixture and the reaction mixture under the wax layer is the specific amplification mixture. Performing a random pre-amplification before the real-time PCR would considerably increase the sensitivity of the specific real-time PCR when testing samples with expected low copy number or precious and irretrievable samples. Performing the two reactions after each other in the same tube minimize the risk for PCR contamination since no transfer of pre-amplified sample is needed. The described method would work on any DNA of any origin, both from non-cellular sources (virus) and from cellular sources (bacteria, archae, eukaryotes), as well as on cDNA.

Inventors:
ERLANDSSON, Lena (Killebäcksvägen 9, Löddeköpinge, S-24632, SE)
FOMSGAARD, Anders (Lundtoftevej 272, Lyngby, DK-2800, DK)
NIELSEN, Lars-Peter (Sophus Bauditz Vej 1A, Lyngby, DK-2800, DK)
Application Number:
DK2011/000026
Publication Date:
October 27, 2011
Filing Date:
April 14, 2011
Export Citation:
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Assignee:
STATENS SERUM INSTITUT (Orestads Boulevard 5, Copenhagen S, DK-2300, DK)
ERLANDSSON, Lena (Killebäcksvägen 9, Löddeköpinge, S-24632, SE)
FOMSGAARD, Anders (Lundtoftevej 272, Lyngby, DK-2800, DK)
NIELSEN, Lars-Peter (Sophus Bauditz Vej 1A, Lyngby, DK-2800, DK)
International Classes:
C12Q1/68
Attorney, Agent or Firm:
TOFT, Lars (Statens Serum Institut, Corporate AffairsOrestads Boulevard 5, Copenhagen S, DK-2300, DK)
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Claims:
CLAIMS

1. A nucleotide sequence amplification method comprising of random unbiased WGA pre- amplification followed by a specific amplification performed in the same tube or vial.

2. A nucleotide sequence amplification method according to claim 1 where the two reaction mixtures initially are separated physically e.g. by a wax-layer, oil-layer or other.

3. A nucleotide sequence amplification method according to claim 2 where the top layer is the pre- amplification reaction mixture and the bottom layer is a specific polymerase chain reaction mixture.

4. A nucleotide sequence amplification method according to claim 3 where the sample is added to the pre-amplification mixture.

5. A nucleotide sequence amplification method according to claim 3 where the pre-amplification mixture is a MDA reaction or a rolling circle reaction

6. A nucleotide sequence amplification method according to claim 5 where the pre-amplification mixture also contain reverse transcription enzyme to generate cDNA from sample RNA and where cDNA is subsequently pre-amplified by an MDA reaction or a rolling circle reaction

7. A nucleotide sequence amplification method according to claim 2 where the pre-amplification mixture are separated from a third mixture containing reverse transcription enzyme separated by wax layers of different melting temperature requirements.

8. A nucleotide sequence amplification method according to any preceding claim, where the specific PCR reaction is chosen from real-time PCR, RT-PCR or multiplex PCR.

9. A nucleotide sequence amplification method according to any preceding claim, where the specific amplification reaction is loop-mediated isothermal amplification of DNA.

10. A nucleotide sequence amplification method according to any preceding claim, where the specific amplification reaction is a NASBA reaction..

11. A nucleotide sequence amplification method according to any preceding claim, where the nucleotide sequence detected are from viruses, bacteria, fungi, parasites or cellular gene sequences or mRNA

12. A nucleotide sequence amplification method according to any preceding claim, where one or more reagents are imbedded inside one or more wax materials and thereby only liberated upon melting of the particular wax.

13. A kit performing the nucleotide amplification method according to claim 1 comprising a tube or vial containing a specific amplification reaction mixture and an unbiased pre-amplification reaction mixture separated by a wax layer.

Description:
New nucleotide sequence amplification method

2. Field of invention

The present invention discloses a nucleotide sequence amplification method comprising a random unbiased whole genome amplification (WGA) pre-amplification reaction followed by a specific amplification performed in the same vial or tube where the two reaction mixtures are separated by a wax layer so the reaction mixture on top of the wax layer is the pre-amplification mixture and the reaction mixture under the wax layer is the specific amplification. A kit for performing the method is also disclosed.

3. General background

Current method of choice for virus identification in many diagnostic laboratories is specific real-time polymerase chain reaction (PCR), where sequence-specific primer pairs are used for each virus, or a group of viruses. This is a rapid, sensitive and specific method that is easy to perform, but has certain limitations. The sensitivity and specificity of any given virus-specific PCR vary depending on the robustness of the primers and the genome variability or mutations in the primer-binding region of the viral genome, as well as the efficiency of the reaction. These limitations of the PCR technique can lead to low or falsely negative results, which in turn may have consequences for the diagnosis and treatment of patients. For example, in a multi- laboratory study of Herpes Simplex virus PCR, which is a widely used and validated PCR assay for cerebrospinal fluids (CSF), it was found that low-level positives were often missed (Schloss et al., 2003).

In some clinical cases the detection level of the routine PCR is not sensitive enough to detect the causing agent. Even if every measure has been taken to design the most sensitive primers and probes from the sequence known about a specific virus, the PCR may still not be sensitive enough for clinical situations with expected low viral load or limited amount of sample. Moreover, there appears to be an inherent limitation to the PCR amplification of small amounts from complex samples, known as the "Monte Carlo effect" (Karrer et al., 1995). Complex samples, such as a clinical sample, containing a low viral copy number close to the detection level of a PCR assay will experience large variations in amplification and reduced reproducibility.

One current technique used to increase the sensitivity of a virus-specific PCR-assay is nested-PCR or semi- nested-PCR, where two sets of primers are used in two successive PCR-runs. The second set of primers, are intended to amplify a target within the amplicon produced in the first run, using the first set of primers. However, the first PCR reaction generates relatively short DNA products that can be difficult to use as templates in the second PCR-reaction. Usually nested-PCR therefore only results in a 10- to 100-fold increase in amplification (Perrott et al., 2009) and present a serious risk of contamination, since amplicons from the first run needs to be transferred to the second run reaction tube. Any transfer of material from one tube containing a PCR amplicon (wheter generated by specific- or random PCR amplification) to another poses a serious risk of PCR product contamination in a routine laboratory and a method that could combine an efficient relatively unbiased pre-amplification of a gene segment larger than a typical diagnostic PCR reaction followed by a shorter specific real-time PCR in one PCR-tube would eliminate that risk and increase the sensitivity of the resulting gene product amplification. Random whole genome amplification (WGA) by isothermal amplification using the Phi29 DNA polymerase has established itself as an excellent alternative to random-PCR based amplification and is described in W09719193 and W09156446, respectively. By isothermal multiple displacement amplification (MDA) in the presence of random primers, Phi29 DNA polymerase allows for uniform amplification across genomes with less than 3-fold bias (Dean et al., 2002; Hosono et al., 2003; Lasken and Egholm, 2003). Furthermore, the Phi29 DNA polymerase has the highest processivity rate reported, -70 000 bases every time it binds (Blanco et al., 1989) and high fidelity with an error rate of only 1 in 10 6 -10 7 bases (Esteban et al., 1993). Furthermore, if presented with 10 ng of high-quality DNA, the Phi29 DNA polymerase can, in a 50μ1 reaction, generate ~40μ of amplified DNA after 16 hours incubation at 30°C (Repli-g Mini/Midi handbook, Qiagen). This amplification is not specific and unbiased as it amplifies all DNA present in the sample.

A pre-amplification of a sample by using WGA prior to a specific PCR could be helpful in clinical cases where the detection limit of the routine real-time PCR assay is not sensitive enough. This could be when only very limited amount of sample material is available or where the pathogen is present at very low amounts and where detection early in a disease progression would benefit the patient with a better prognostic outcome. For example this is true for cases of suspected progressive multifocal leukoencephalopathy (PML) in the central nervous system, where an early correct detection of the causing agent JC polyomavirus in the spinal fluid increases the chances for survival (Landry et al., 2008; Linda et al., 2009). Even if the real-time PCR used for a specific virus is very sensitive, it is always a risk for false negative results when analysing samples with pathogens that are at the detection limit of the assay. In this case a nonbiased pre-amplification would be helpful to bring up the copy number in the sample in a form suitable for PCR amplification before the virus-specific real-time PCR assay is run, thereby giving the possibility for an ultrasensitive and an earlier detection of disease-causing viruses in clinical samples.

Also, in situations where the sample is precious and irretrievable, a pre-amplification before a specific PCR would be helpful to reduce the amount of sample needed for an analysis. This could be dried blood samples on Guthrie cards from routine neonatal screening, where only a small piece of filter paper can be used for DNA extraction. In children with suspected congenital Cytomegalovirus (CMV) infection, with the most common symptom being sensory neural hearing loss (SNHL), the dried blood samples are revisited to establish the presence of CMV at birth (Barbi et al., 2000; de Vries et al., 2009). In cases like this, a random nonbiased pre-amplification of purified DNA would help gain more material for a more accurate analysis testing. Such methods are not limited to detection of virus DNA but could be other target DNA and/or RNA-

Similar situations where the amount of material is limiting and contamination needs to be avoided could be when working with ancient samples, forensic samples or single-cells from culture or cell-sorting. It would also be possible to combine the described method with a suitable reverse transcription (RT)-step, thereby enabling pre-amplification plus specific real-time PCR of mRNA to look at expression patterns or of viral RNA-genomes. To be able to amplify the signal in this way would help in the monitoring of low viral load seen in situations as in certain HTV-infections for example so called "elite controllers" or patients in antiretroviral therapy. Furthermore, the pre-amplification could also be combined with a multiplex real-time PCR reaction detecting multiple pathogens, instead of a singleplex reaction as used here.

It would therefore be useful with a method that, in one tube, combines a pre-amplification with a specific PCR, thereby moving the sample-copy number away from the assay's detection limit and increasing the sensitivity and over-all reproducibility of the assay. Combining a pre-amplification with a specific real-time PCR in one PCR-tube would also eliminate the risk of contamination since no transfer of pre-amplified material is needed.

4. Summary of the invention

The described method discloses a technique that, in one tube, combines a random pre-amplification (nonbiased isothermal WGA) reaction with a specific real-time PCR, performed one after the other.

Performing a random pre-amplification before the real-time PCR would considerably increase the sensitivity of the specific real-time PCR when testing samples with expected low copy number or precious and irretrievable limits of samples. Performing the two reactions after each other in the same tube minimize the risk for PCR contamination since no transfer of pre-amplified sample is needed. We also show that it can increase the signal-intensity of a specific real-time PCR at least 100xl0 6 -fold and that the pre-amplification can make detection possible of samples normally under the detection level of a specific real-time PCR (Figure 3B). This would be useful when testing samples with expected low copy number or precious and irretrievable samples such as forensic samples. The described method would work on any DNA of any origin, both from non-cellular sources (virus) and from cellular sources (bacteria, archae, eukaryotes), as well as on cDNA of sizes >2kb (Berthet et al., 2008) (Data not shown) (Repli-g Mini/Midi handbook, Qiagen).

5. Detailed description of the invention

The present invention discloses a nucleotide amplification method comprising of random unbiased whole genome amplification (WGA) pre-amplification followed by a specific amplification performed in the same vial or tube. The two reaction mixtures are separated by a wax layer so the reaction mixture on top of the wax layer is the pre-amplification mixture and the reaction mixture under the wax layer is the specific amplification.

In a preferred embodiment the WGA pre-amplification reaction is a multiple displacement amplification (MDA) or a rolling circle amplification and the specific amplification is a polymerase chain reaction (PCR).

The PCR mixture comprises primers and probes for detecting a specific DNA or RNA of any origin, both from non-cellular sources (virus) and from cellular sources (bacteria, archae, eukaryotes), as well as cDNA.

In another embodiment the invention discloses a kit for performing said PCR method comprising a vial or tube containing the two reaction mixtures separated by a wax layer.

Definitions:

PCR: Polymerase chain reaction (PCR) refers to all techniques that amplifies a single or few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase (after which the method is named) are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. The PCR application employs a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermits aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary first to physically separate the two strands in a DNA double helix at a high temperature in a process called DNA melting. At a lower temperature, each strand is then used as the template in DNA synthesis by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

A lot of variants of PCR techniques are known e.g. Allele-specific PCR, Polymerase Cycling Assembly (PCA), Asymmetric PCR, Helicase-dependent amplification, Hot-start PCR, Intersequence-specific PCR (ISSR), Ligation-mediated PCR, Methylation-specific PCR (MSP), Multiplex Ligation-dependent Probe Amplification (MLPA), Multiplex-PCR, Nested PCR, Overlap-extension PCR, Quantitative PCR (Q-PCR) also known as Real-time PCR and not to be mixed up with Reverse Transcription PCR (RT-PCR). All these PCR methods will be well known to the skilled worker.

Specific PCR product: refers to a method that generates a single specific PCR product with the sequence and size predicted from the sequences of the primers and the region of nucleic acid to which the primer or probe were designed to anneal. The specific PCR product can be detected in various ways known to the skilled worker or analyzed by sequencing.

WGA: Whole Genome Amplification refers to an in vitro method that is used to amplify a genomic DNA sample, and generate large amounts of amplified DNA for further molecular genetic analyses. Different methods have been developed to amplify the whole genome, including primer extension pre-amplification (PEP), degenerate oligonucleotide primed PCR (DOP-PCR) and multiple displacement amplification (MDA).

MDA: Multiple displacement amplification is a non-PCR based DNA amplification method. It is an isothermal method that preferentially utilizes bacteriophage phi29 DNA polymerase (Dean et al., 2002) and random hexamers for amplification (Dean et al., 2001). The reaction starts by the annealing of random hexamer primers all over a single-stranded DNA template followed by polymerization by Phi29 DNA polymerase at each of these annealing sites. When the polymerase reaches another annealing site downstream, instead of halting, it displaces the newly produced DNA strand and continues its own strand elongation. In this way, every polymerase generates long strands of newly synthesized DNA, and further primer annealing and strand displacement on these newly synthesized templates results in a hyper-branched DNA network. This results in very high yields and high quality products.

Rolling circle amplification fRCA): refers to prolonged extension of an oligonucleotide primer annealed to a circular template DNA producing a continuous sequence of tandem copies of the circle (a concatemer).

Through multiple displacement amplification (MDA) a cascade of strand displacement reactions results in an exponential amplification. In RCA, one or several primers can be used, which can be either specific or random. Phi29 polymerase: is a DNA polymerase from the phage Phi29 with unique properties. By isothermal MDA in the presence of random primers, Phi29 DNA polymerase allows for uniform amplification across genomes with less than 3-fold bias (Dean et al., 2002; Hosono et al., 2003; Lasken and Egholm, 2003). Furthermore, the Phi29 DNA polymerase has the highest processivity rate reported, -70 000 bases every time it binds (Blanco et al., 1989) and high fidelity with an error rate of only 1 in 10 6 -10 7 bases (Esteban et al., 1993).

Repli-g: A commercially available kit from Qiagen for WGA by MDA through the used of Phi29 DNA polymerase and random hexamer primers. According to the Repli-g Midi kit-protocol (Qiagen), the reaction should be run for 16 hours to reach maximum yield of amplified DNA (~40μg in 50μ1 when starting with lOng, Qiagen). As for any Phi29 DNA polymerase-reaction, smaller fragments are inefficiently amplified by the Phi29 DNA polymerase (Berthet et al., 2008) (Repli-g Mini/Midi handbook, Qiagen).

Tube for PCR reactions: refers to any container suitable for holding PCR reagents and test sample during PCR amplifications. The container will have a tightly fitting lid which blocks vapour escape and contamination.

Wax: include greases and refers to an organic substance, solid at temperatures below about 40°C, which melts at somewhat higher temperatures to form a liquid with a lower density than water. Waxes tend to adhere to solids thereby sealing and underlying water layer when placed together in a tube. Typical waxes are a mixture of high molecular weight hydrocarbons (greases) and as pure compounds eicosane C20H42, octacosane C28H180O8, cetyl palmitate C32H64o2 and pentaerythritol tetrabehenate C93H180O8. Typical useful wax mixtures include paraffin, Paraplast (trade name of Sherwood Medical), Ultraflex (trade name of Petrolite Corporation), BeSquarel 75 (trade name of Petrolite Corporation) and Ampliwax (trade name of Applied Biosystems). Waxes can be prepared by mixing pure or mixed waxes with one another or with greases or oils in any ratios which preserve the relative hardness and stickiness characteristic of a wax. The present invention discloses a method that, in one tube, combines random nonbiased pre-amplification with a specific PCR, preferably a real-time PCR. The pre-amplification (MDA by Phi29 DNA polymerase) is run before the specific PCR in the same tube. The two reactions are separated from each other by a wax- layer. Combining the two reactions in one tube removes the need for transfer of pre-amplified material to the PCR-reaction and the risk of contamination between samples and from the environment. Any transfer of material from one tube to another poses a serious risk of contamination in a routine laboratory.

We wanted to do a random unbiased isothermal pre-amplification of the sample before the specific real-time PCR. Furthermore, we wanted to combine the two reactions in one tube, where the pre-amplification step was run first, followed by the specific PCR reaction. To achieve this, we separated the two reactions physically by melting a gem of wax in-between the two layers (Figure 1), so that the PCR-master mix was locked in the bottom of the tube and separated from the pre-amplification reaction that was placed on top of the wax-seal. In this way, after adding the sample to the top, the pre-amplification could be performed first, at 30°C for 16 hours, while separated from the PCR-reaction mix. By incubating for 16 hours, the pre- amplification is run to completion to avoid any surplus of oligonucleotides that can interfere with the following specific PCR reaction. Furthermore, the Hot Start Taq polymerase present in the PCR reaction mix is inactive at 30°C. After completed pre-amplification, 15-minutes incubation at 95°C inactivated the Phi29 DNA polymerase, activated the Hot Start Taq polymerase and melted the wax so that the two reactions could be mixed. Thereafter the virus-specific real-time PCR-reaction was run for 45 cycles. All steps are performed after each other in a thermal PCR cycler.

We show that the described method can increase the signal-intensity of a specific real-time PCR at least 100xl0 6 -fold and that the pre-amplification can make detection possible of samples normally under the detection level of a specific real-time PCR (Figure 3B). This would be useful when testing samples with expected low copy number or precious and irretrievable samples such as forensic samples. The described method would work on any nucleic acid of any origin, both from non-cellular sources (virus) and from cellular sources (bacteria, archae, eukaryotes), as well as on cDNA of sizes >2kb (Berthet et al., 2008) (Repli-g Mini/Midi handbook, Qiagen).

Depending on the type of sample to be analysed, various nucleic acid (NA)-purification methods or protocols can be applied, known to the skilled worker. Also, depending on the type of sample, NA can be purified from pelleted sample or from supernatants depending on where the NA of interest is situated in the sample. If analysing tissue samples, they need to be homogenized prior to NA purification. Liquid clinical samples, such as cerebrospinal fluid (CSF), urine and serum, are normally first centrifuged and the resulting supernatant used for NA purification. Other clinical samples, such as various swabs, are usually first transferred into a suitable liquid prior to centrifugation and the resulting supernatant used for NA purification. A number of commercially available kits can be used for the NA purification step. It is recommended that in all cases where Phi29 DNA polymerase-amplification is used down-stream of the NA purification step, carrierRNA should not be included in the purification process since it seems to interfere with the downstream amplification (Repli-g Midi handbook, Qiagen). The extracted viral NA should be stored at -20°C or immediately used.

The pre-amplification used here is preferably a random isothermal MDA-reaction performed by the Pni29 DNA polymerase together with random hexamer primers, but other isothermal amplification-reactions, e.g. primase-based WGA (pWGA; Rapisome, BioHelix Corp.) could also work in this set-up. Various commercial kits utilizing the Phi29 DNA polymerase is available for purchase, we decided to use Repli-g from Qiagen but others would work equally well. The concentration of the Phi29 DNA polymerase and the other ingredients such as primers, nucleotides and buffer is only known to the manufacturer.

The pre-amplification reaction is set up in a total volume of ΙΟμΙ and the reaction contains, in addition to the polymerase and the random primers, also nucleotides and a suitable buffer that supports the polymerase. To find a good balance between the volumes of the Repli-g reaction and the real-time PCR reaction, we tested different ratios (1 :2, 1 :3, 1 :4 and 1 :5) and found a ratio of 1 :3, with ΙΟμΙ of Repli-g reaction and 30μ1 of PCR-reaction, to work well. A volume of one microliter containing the DNA is added to the pre- amplification reaction (Repli-g). The sample DNA needs to be single-stranded, which in the Repli-g kit is achieved by the use of chemical denaturation described in the Repli-g protocol. Other kits use heat- denaturation at 95°C, which is also a suitable method. The reaction is isothermal and performed at 30°C. We perform the pre-amplification reaction for 16 hours, so that when working with samples containing few DNA-copies the pre-amplification is run to completion to avoid any surplus of oligonucleotides that can interfere with the following specific PCR reaction. Samples containing high amounts of DNA will reach completion in far less time than 16 hours, but those are not the kind of samples in need of a pre-amplification such as the described method.

The pre-amplification is not specific; any suitable DNA present in the reaction will be amplified

To create a wax-layer separating the pre-amplification reaction and the real-time PCR reaction, we used the commercially available AmpliWax (Applied Biosystems), but other types of waxes would work equally well. After the real-time PCR reaction (volume 30μ1) has been added to a 0.2ml PCR-tube, a wax pellet is added on top. The tube is put in a heater (65°C) for a couple of minutes to melt the wax on top of the aqueous real- time PCR reaction and thereafter cooled to room temperature to solidify. In this way the wax is placed as a seal on top of the real-time PCR reaction, allowing for the addition of another aqueous reaction mixture on top of the wax-layer. The real-time PCR reaction performed after the random pre-amplification can be a multi-plex or single-plex specific PCR. This means that it generates one or several specific PCR products with the sequence and size predicted from the sequences of the primers/probe and the region of DNA to which they were designed to anneal to. The pre-amplification and the real-time PCR are combined in the same tube, but separated physically by a wax-layer. The tube is placed in a thermo cycler and the pre-amplification performed first, followed by a heating step and then the real-time PCR reaction for a set number of cycles, typically 40-45. The thermo cycler detects the fluorescence generated during each of the PCR cycles, which is then analysed by the machines software to generate curves and Ct-values to detect the DNA present in the sample.

The pre-amplification is not specific and amplifies any DNA present in the sample, while the real-time PCR reaction is specific. DNA of any origin can be analysed in a real-time PCR reaction, as long as there is enough known sequence to make it possible to design specific primers and probe for a region within the DNA-fragment. The origin of the DNA can be both non-cellular (viral) and cellular (bacterial, archae, eukaryotic).

When performing the described method on samples with expected low copy number and at the detection limit for any specific real-time PCR, it would be recommended to run samples in duplicates or even triplicates. This method has eliminated the risk of cross-contamination between samples when using a pre-amplification step prior to a specific PCR amplification, since no transfer of pre-amplified material is needed. Since the pre-amplification and the specific PCR-reaction are run in the same tube, less time is spent on pipetting making the method less labour intense. The method can be run overnight and thereby fitted well into routine schedules.

The described method could be helpful in clinical cases where the detection limit of the routine real-time PCR assay is not sensitive enough. This could be when the virus is present at very low amounts and where detection early in a disease progression would benefit the patient with a better prognostic outcome. This is true for cases of suspected progressive multifocal leukoencephalopathy (PML) in the central nervous system, where an early correct detection of the causing agent JC polyomavirus in the spinal fluid increases the chances for survival (Landry et al., 2008; Linda et al., 2009).

Even if the real-time PCR used for a specific virus is very sensitive, it is always a risk for false negative results when analysing samples that are at the detection limit of the assay. For example, in a multi-laboratory study of Herpes Simplex virus PCR, which is a widely used and validated PCR assay for cerebrospinal fluids (CSF), it was found that low-level positives were often missed (Schloss et al., 2003). In this case a nonbiased pre-amplification would be helpful to bring up the copy number in the sample before the virus-specific realtime PCR assay is run, thereby giving the possibility of accurately detecting the low-level positives in clinical samples.

Also, in situations where the sample is precious and irretrievable, a pre-amplification before a specific PCR would be helpful to reduce the amount of sample needed for an analysis. This could be dried blood samples on Guthrie cards from routine neonatal screening, where only a small piece of filter paper can be used for DNA extraction. In children with suspected congenital Cytomegalovirus (CMV) infection, with the most common symptom being sensori neural hearing loss (SNHL), the dried blood samples are revisited to establish the presence of CMV at birth (Barbi et al., 2000; de Vries et al., 2009). In cases like this, a random nonbiased pre-amplification of purified DNA would help gain more material for a more accurate analysis. Similar situations where the amount of material is limiting and contamination needs to be avoided could be when working with ancient samples, forensic samples or single-cells from culture or cell-sorting. Forensic samples, where only small amounts of semen, blood, hair follicles or cells may be available for DNA analyses, could benefit from a pre-amplification of the human genomic DNA before specific PCR-analysis. It would also be possible to combine the described method with a suitable reverse transcription (RT)-step, thereby enabling pre-amplification plus specific real-time PCR of mRNA to look at expression patterns or of viral RNA-genomes. To be able to amplify the signal in this way would help in the monitoring of low viral load seen in situations as in HIV-infections. The pre-amplification could also be combined with a multiplex real-time PCR reaction, instead of a singleplex reaction as used here.

Torque Teno virus (1 TV) appears to be ubiquitous in humans, elicits seemingly innocuous infections and does not exhibit seasonal fluctuations or epidemic spikes. TTV is transmitted primarily via the fecal-oral route and has been tested as an appropriate indicator of viral pathogens in drinking water (Griffin et al., 2008). TTV is a small circular DNA virus and is easily amplified by the use of MDA using the Phi29 DNA polymerase, thereby being a suitable candidate for the use of a combined pre-amplification and specific realtime PCR method to analyse drinking water quality around the world. Due to the increased sensitivity, the described method is predicted to have significant value and impact when screening blood and blood components for viruses such as hepatitis B, hepatitis C and human

immunodeficiency virus (HIV).

The Phi29 DNA polymerase does not efficiently amplify DNA fragments of less than 2kb in size (Berthet et al., 2008) (Repli-g Mini/Midi handbook, Qiagen), but works well on DNA of any origin that is 2kb or larger. The sensitivity and specificity of any given specific PCR vary depending on the robustness of the primers and the genome variability or mutations in the primer-binding region of the DNA fragment being analysed, as well as the efficiency of the reaction. A detection limit with maintained reproducibility at 10 copies per real-time PCR reaction, have been reported for a JC polyomavirus-specific real-time PCR(Ryschkewitsch et al., 2004). With the additional use of a pre-amplification before the specific real-time PCR, a single copy of DNA should theoretically, with high reproducibility, be detected. In Figure 3 we show that a diluted JC polyomavirus-positive sample could only be detected when combining a pre-amplification with the specific real-time PCR.

Figure legends

Figure 1. A. Schematic picture showing how the AmpliWax works as a physical barrier to separate the random pre-amplification reaction (Repli-g) and the specific real-time PCR reaction. B. AmpliWax does not interfere with the JC-specific real-time PCR reaction.

Figure 2. Combination of 8 hours of random nonbiased Phi29-amplification with JC-specific real-time PCR in one PCR-tube results in a 17.6 million-fold increase in signal (ACt=24.07).

Figure 3. A. Real-time PCR only on single sample of 100-fold and duplicate samples of 6400-fold diluted JC viral DNA. B. Sixteen hours of Repli-g amplification + real-time PCR on single sample of 100-fold and duplicate samples of 6400-fold diluted JC viral DNA.

Figure 4. A. Sixteen hours of Repli-g amplification + real-time PCR on HPV16-positive clinical sample. B Sixteen hours of Repli-g amplification followed by a HSV1 -specific real-time PCR on a HSV1 -positive clinical sample. EXAMPLES

Example 1: Detection of the circular 5kb-large double-stranded DNA genome of the JC polvomavirus Virus sample

Samples used were JC polyomavirus-positive cerebrospinal fluid (CSF) received for routine diagnostic analysis at the Department of Virology, Statens Serum Institut, Copenhagen, Denmark (accredited and quality-controlled Danish National reference diagnostic routine laboratory (ISO 17025); www.ssi.dk). The JC polyomavirus is a circular double-stranded DNA virus of 5kb, which may cause fatal progressive multifocal leukoencephalopathy (PML) in immunodeficient or immunosuppressed patients (Padgett et al., 1971).

Pre-treatment and extraction of CSF samples

Two hundred microliters of CSF was transferred to an eppendorf tube and centrifuged at 17 000 g for 10 minutes to remove cell debris. The supernatant was then filtered through a 0.22μπι Ultrafree MC spin filter (Millipore) at 2000 g for 2 minutes and thereafter DNase treated (6U DNasel Amplification grade,

Invitrogen) for 1 ½ hours at room temperature while shaken in a Thermomixer (AH Diagnostics). The viral nucleic acid (NA) was extracted using the PureLink Viral RNA DNA kit (Invitrogen), without the addition of carrier RNA. The extracted viral NA was eluted with 20-30μ1 DNase/RNase-free sterile water, and stored at -20°C or immediately used.

Combining pre-amplification with a JC virus-specific PCR

Real-time PCR reaction mix specific for JC virus was prepared, either using described primers and probe (MacKenzie et al., 2003; Ryschkewitsch et al., 2004) or primers and probe from an in-house real-time PCR assay. Described primers and internal probe were: JCT-1 (5 ' -AGA GTG TTG GGA TCC TGT GTT TT-3 '; SEQ ID NO 1 ), JCT-2 (5 '-GAG AAG TGG GAT GAA GAC CTG TTT-3 ' ; SEQ ID NO 2) and JCT-1.1 (5 - FAM-TCA TCA CTG GCA AAC ATT TCT TCA TGG C-TAMRA-3 SEQ ID NO 3) where FAM is the fluorescence reporter dye fluorescein and TAMRA is the fluorescence quencher dye tetramethylrhociarnine. In-house PCR primers and internal probe were: JC-F (5'-TGA ACC AAA AGC TAC ATA GGT AAG TAA TG-3'; SEQ ID NO 4), JC-R (5'-AAT CCT GTG GCA GCA G-3'; SEQ ID NO 5) and JC-P (5'-FAM-TTC ATG GGT GCC GCA CTT GCA-BHQl-3'; SEQ ID NO 6) where BHQl is the fluorescent dye "black hole quencher-1". Thirty microliters of PCR reaction mix containing 15μ1 of 2x QuantiTect Multiplex PCR NoROX Master mix (contains HotStarTaq DNA polymerase, Qiagen), 500nM of each primer and ΙΟΟηΜ probe was put in a 0,2ml PCR-micro tube. One pellet of AmpliWax PCR Gem 50 (PE Applied Biosystems) was added on top of the PCR reaction mix, the tube put in a cycler and incubated at 60°C for 5 min to melt the wax followed by cooling at 37°C to solidify the wax on top of the PCR reaction mix. Next, the random Phi29-amplification reaction was prepared by making a ΙΟμΙ-Repli-g Midi reaction (only 1/5 of the normal reaction) according to manufacturer's protocol (Repli-g Midi kit, Qiagen). Shortly, Ιμΐ of purified viral DNA was mixed with Ιμΐ of denaturation-solution and incubated at room temperature for 3 minutes. Two microliters of stop solution was added and the sample mixed. Thereafter, 5.8μ1 of Repli-g Midi reaction buffer and 0.2μ1 of Phi29 DNA polymerase was added and the sample vortexed. After a short centrifugation, the Repli-g reaction was added on top of the solidified AmpliWax, the tube closed and put in a thermal cycler (Mx3005P, Stratagene). The following program was run: 30°C for 16 hours to run the Repli-g reaction; 95°C for 15 minutes to inactivate the Phi29 DNA polymerase, to activate the Taq polymerase and to melt the wax so that the Repli-g product was mixed with the PCR reaction mix; followed by a 45 cycle-real- time PCR with 95°C for 15 seconds and 60°C for 1 minutes. The assay was performed in an Mx3005P-cycler (Stratagene).

Results

To test if the Ampli Wax-seal would interfere with the real-time PCR reaction, we took Ιμΐ of purified JC- DNA from a CSF clinical sample and added to a JC-specific real-time PCR reaction mix. A pellet of AmpliWax was melted on top and the real-time PCR performed (Figure IB). We saw no difference in PCR- performance when comparing a reaction containing AmpliWax with a reaction without AmpliWax, and therefore concluded that the AmpliWax-seal did not interfere with the JC-specific real-time PCR.

To find a good balance between the volumes of the Repli-g reaction and the real-time PCR reaction, we tested different ratios and found a ratio of 1:3, with ΙΟμΙ of Repli-g reaction and 30μ1 of PCR-reaction, to work well (data not shown). Using this ratio we combined 8 hours of Repli-g Midi reaction with the AmpliWax-sealed JC-specific real-time PCR-reaction (Figure 2). The sample used was a 50-fold diluted purified JC-DNA from a CSF clinical sample. In parallel as a control, the sample in just water was added on top of a wax-sealed PCR reaction mix (PCR only). The Repli-g reaction amplified the JC-positive CSF- sample 17.6xl0 6 -fold (ΔΟ=24.07) compared to the sample that only went through the PCR-step.

To test the sensitivity on a clinical sample, we used purified JC-DNA from a CSF clinical sample. We diluted the JC-DNA 100- and 6400-fold. A 16 hour Repli-g reaction followed by JC-specific real-time PCR was performed (Figure 3). The 100-fold diluted sample resulted in a very strong JC-signal (Ct= ~4) after the combined amplification and PCR, which resulted in a 268x10 6 -fold increase in signal (ACt= 28) compared to PCR only. Neither of the duplicate samples from the 6400-fold diluted JC-sample gave any signal in PCR only (Figure 3 A). However, one of them gave a signal after combining pre-amplification with the JC-specific PCR (Ct= 27) while the other one did not (Figure 3B) demonstrating that samples under the detection limit of the real-time PCR assay may get amplified and show a good signal in our combined pre-amplification + PCR-assay.

We found that samples with high copy number of viral genomes exhausted the reagents in less than 16 hours, thereby completing the reaction. These samples generated an access of products that looked to be inhibitory in the downstream PCR-reaction, as seen with the wave-shaped curve after real-time PCR in Figure 3B (100- fold diluted sample). If the pre-amplification had been run in a separate tube, as with nested-PCR, one would only transfer minute amounts of the product to the real-time PCR master mix to not over-saturate the reaction. But since the reactions now appear in the same tube, all generated product is transferred to the real- time PCR master mix when the wax is melted. This does not really pose a problem since these curves are easily distinguished as positive samples, and would only occur when high-copy number samples are analysed. When samples with low copy number were used, the full reaction time of 16 hours was needed for the reaction to run out of reagents or to reach a point where the level of non-used random primers were low enough to not interfere with the downstream real-time PCR (6400-fold diluted sample in Figure 3B).

Example 2: Detection of the circular 8kb-large double-stranded DNA genome of the HPV-16 and the linear 152kb-large double-stranded DNA genome of the HSV1

Virus sample

Samples used were HPV-16-positive cervix smears and HSV-1 -positive swabs received for routine diagnostic analysis at the Department of Virology, Statens Serum Institut, Copenhagen, Denmark (accredited and quality-controlled Danish National reference diagnostic routine laboratory (ISO 17025); www.ssi.dk). The Human papilloma virus-16 (HPV-16) is a circular double-stranded DNA virus of 8kb, which may cause cervical cancer in women. Human herpes simplex-1 virus is a linear double-stranded DNA virus of 152kb.

Pre-treatment and extraction of viral nucleic acid

Two hundred microliters of sample was transferred to an eppendorf tube and centrifuged at 17 000 g for 10 minutes to remove cell debris. The supernatant was then filtered through a 0.22μπι Ultrafree MC spin filter (Millipore) at 2000 g for 2 minutes and thereafter DNase treated (6U DNasel Amplification grade,

Invitrogen) for 1 ½ hours at room temperature while shaken in a Thermomixer (AH Diagnostics). The viral nucleic acid (NA) was extracted using the PureLink Viral RNA/DNA kit (Invitrogen), without the addition of carrier RNA. The extracted viral NA was eluted with 20-30μ1 DNase RNase-free sterile water, and stored at -20°C or immediately used. Combining pre-ampliflcation with a HPV-16- or HSV-l-virus-specific PCR

Real-time PCR reaction mix specific for HPV-16 virus was prepared, using primers and probe from an in- house real-time PCR assay. In-house primers and internal probe were: HPV16-E6-12F (5'- CGA CCC AGA AAG TTA CCA CAG TT-3'; SEQ ID NO 7), HPV16-E6-12R (5'- TGT TGC TTG CAG TAC ACA CAT TCT A-3'; SEQ ID NO 8) and HPV16-E6-12P (5'-FAM- CAC AGA GCT GCA AAC AAC TAT ACA TGA TAT AAT-BHQ I-3'; SEQ ID NO 9). Real-time PCR reaction mix specific for HSV-1 virus was prepared, using primers and probe from an in-house real-time PCR assay. In-house primers and internal probe were: HSVl -LPs (5'-TGT GGT GTT TTT GGC ATC AT-3'; SEQ ID NO 10), HSVl-LPas (5'- CCG ACA AGA ACC AAA AGG AA -3'; SEQ ID NO 1 1) and HSVl-LPprobe (5'-FAM- CAT GCG TGC CGT TGT TCC CA-BHQ1-3'; SEQ ID NO 12). Thirty microliters of PCR reaction mix containing 15μ1 of 2x QuantiTect Multiplex PCR NoROX Master mix (contains HotStarTaq DNA polymerase, Qiagen), 500nM of each primer and lOOnM probe was put in a 0,2ml PCR-micro tube. One pellet of AmpliWax PCR Gem 50 (PE Applied Biosystems) was added on top of the PCR reaction mix, the tube put in a cycler and incubated at 60°C for 5 min to melt the wax followed by cooling at 37°C to solidify the wax on top of the PCR reaction mix. Next, the random Phi29-amplification reaction was prepared by making a ΙΟμΙ-Repli-g Midi reaction (only 1/5 of the normal reaction) according to manufacturer's protocol (Repli-g Midi kit, Qiagen). Shortly, Ι μΐ of purified viral DNA was mixed with Ιμΐ of denaturation-solution and incubated at room temperature for 3 minutes. Two microliters of stop solution was added and the sample mixed. Thereafter, 5.8μ1 of Repli-g Midi reaction buffer and 0.2μ1 of Phi29 DNA polymerase was added and the sample vortexed. After a short centrifugation, the Repli-g reaction was added on top of the solidified AmpliWax, the tube closed and put in a thermal cycler (Mx3005P, Stratagene). The following program was run: 30°C for 16 hours to run the Repli- g reaction; 95°C for 15 minutes to inactivate the Phi29 DNA polymerase, to activate the Taq polymerase and to melt the wax so that the Repli-g product was mixed with the PCR reaction mix; followed by a 45 cycle- real-time PCR with 95°C for 15 seconds and 60°C for I minutes. The assay was performed in an Mx3005P- cycler (Stratagene).

Results

To show that this method should work equally well with other viral DNA samples, we tested it on purified HPV-16 and HSV-1 viral DNA (Figure 4). Combining a Repli-g pre-amplification with a HPV-16 specific- PCR resulted in a 4300xl0 6 -fold increase in signal (ACt=32) compared to PCR only (Figure 4A). Combining the pre-amplification with a HSV-1 -specific PCR resulted in a 134xl0 6 -fold increase in signal (ACt=27) compared to PCR only (Figure 4B).

We show this method to work with two different circular and one linear double-stranded viral DNA genomes, but it would work equally well on any circular or linear double- or single-stranded DNA of any origin. However, they should be of a size of 2kb or larger for the Phi29 DNA polymerase amplification to work properly (Berthet et al., 2008) (Data not shown) (Repli-g Mini/Midi handbook, Qiagen).

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