FIELD OF THE INVENTION Embodiments described here concern methods to prepare a crude biological sample in order to make it directly available for molecular biology reactions with the particular purpose of detecting and quantifying the presence of RNA, that is, for RNA analysis directly in a crude biological sample, preventing the degradation of the RNA itself. Other embodiments also concern kits for preparing a biological sample for the purpose of RNA analysis directly in a crude biological sample.

BACKGROUND OF THE INVENTION

It is known to use the RNA of pathogenic microorganisms, viruses in particular, as a biomarker to certify their presence in biological samples for the diagnosis, or assistance in the diagnosis, of particular infectious pathological states. RNA can be detected by nucleic acid amplification systems such as RT-PCR (Reverse Transcriptase - Polymerase Chain Reaction), a reaction which provides an initial isothermal reaction where the RNA is copied into a DNA strand which will then be exponentially amplified in many copies of DNA or by instruments that provide hybridization steps of the target RNA, with probes able to recognize such targets, followed by incubations with elements that can recognize the hybridized targets and give a signal mediated by an enzymatic reaction detectable in luminescence or fluorescence or other. These RNA targets, to be detected effectively, must often be purified and isolated from the rest of the biological material contained in samples such as saliva, sputum, expectorate, vaginal mucus, bronchial mucus, nasal mucus, oropharyngeal mucus, urine, feces, whole blood, blood serum, skin secretions, spinal fluid, pulmonary and alveolar secretions, cervicovaginal samples and tissue. These purification steps of the RNA allow to remove agents that can inhibit PCR reactions, concentrate RNA, and remove agents able to degrade the RNA. These steps often represent a source of variability, they lengthen the detection and analysis times and entail the use of numerous reagents which impact on the costs of the process.

Among the known technologies for the purification of RNA it is known to use systems based on solvents and columns with high affinity for nucleic acids. Providing methods that make it possible to make the biological sample directly available for reactions able to detect the presence of RNA without using purification steps would allow to reduce diagnostic response times and reduce analysis costs. Known methods to treat the crude sample in order to make RNA available are often not very effective, since RNA is a very fragile molecule and subject to degradation. The methods used to treat the crude sample in the pre- analytical step in order to make it available for RNA analysis are all inefficient when compared to the classic nucleic acid extraction, both in ion exchange resin columns and also with phenols, since RNA is a fragile molecule and easily subject to degradation. Furthermore, avoiding the extraction of RNA for the analysis of the biological material for diagnostic purposes could effectively counter the problems related to the difficult procurement of reagents for the extraction of nucleic acids, in particular during times of high pressure for laboratories and the health system in general, as was highlighted during the SARS-CoV-2 virus pandemic.

There is therefore a need to perfect a method to prepare a biological sample for RNA analysis directly in a crude biological sample which can overcome at least one of the disadvantages of the state of the art.

In particular, one purpose of the present invention is to provide methods and kits for preparing a biological sample to allow to detect integral RNA directly from crude biological samples, preventing the degradation of the RNA itself.

The Applicant has devised, tested and embodied the present invention to overcome the shortcomings of the state of the art and to obtain these and other purposes and advantages. SUMMARY OF THE INVENTION

The present invention is set forth and characterized in the independent claims. The dependent claims describe other characteristics of the present invention or variants to the main inventive idea.

In accordance with the above purposes, a method is provided to prepare a crude biological sample which overcomes the limits of the state of the art and eliminates the defects present therein. The method described here, in particular, provides to prepare a biological sample for RNA analysis directly in a crude biological sample, preventing the degradation of the RNA itself. In some embodiments described here, in particular, the method can be provided to process biological samples such as saliva, vaginal mucus, bronchial mucus, nasal mucus, oropharyngeal mucus, urine, feces, whole blood, blood serum, skin secretions, spinal fluid, pulmonary secretions, alveolar secretions, cervicovaginal samples and tissue containing RNA in order to make them compatible with RNA detection systems, for example based on RT-PCR or hybridization with probes and subsequent amplification of the signal detectable in luminescence, fluorescence, amperometry, voltammetry or impedance. Some embodiments of the method described here, in particular, can comprise a series of heat treatment steps at different temperatures, they can include the presence of one or several proteolytic enzymes and organic and/or inorganic RNases inhibitors, RNA degrading enzymes present in the biological samples.

In some embodiments, the method described here comprises:

- supplying a crude biological sample in liquid form or resuspended in an aqueous solution free of protein denaturing substances,

- combining the crude sample with a lysis solution containing one or several proteolytic enzymes,

- subjecting the crude biological sample combined with said lysis solution to a heat treatment (or first heat treatment) at a temperature between 25°C and 85°C for a time between 1 and 25 minutes,

- subjecting at least part of the biological sample combined with the lysis solution of the previous step to an additional heat treatment (or second or last heat treatment) at a temperature between 70°C and 250°C for a time between 3 seconds and 45 minutes. In some embodiments, in particular, the method as above can provide to resuspend or dilute the crude biological sample in a lysis buffer containing said one or several proteolytic enzymes.

The one or several proteolytic enzymes can for example be natural or recombinant. According to one possible aspect of the invention, the crude biological sample can be in dried form prior to the resuspension in the aqueous solution free of protein denaturing substances.

According to some embodiments, it can be provided to dilute the crude biological sample, before subjecting it to the first heat treatment, with an aqueous solution free of protein denaturing substances.

Some embodiments can provide that the one or several proteolytic enzymes, either natural or recombinant, are selected from a group consisting of: trypsin, furin, proteinase K, endoproteinase, elastase, streptogrisin, thrombin, pepsin, chymostrypsin, papain, bromelain, pancreatin, amylase, cysteine protease, aspartate protease, carboxy protease. One, two, three or even more of the proteolytic enzymes mentioned above can be used in the lysis solution.

Preferred embodiments can provide that the lysis solution contains at least proteinase K and trypsin, in particular between 0.1 pg/mL and 10 mg/mL of each enzyme, for example.

The denaturing substances as above can be acids, alkalis, heavy metal salts, ethanol, guanidine detergents, guanidinium chloride, guanidinium thiocyanate, urea, formamide, guanidine, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, ammonium thiocyanate, phenol.

The method according to the present invention is able to make the sample suitable to be used in subsequent RNA detection methods, such as RT-PCR, which make use of both fluorescent probes based on Taqman, Scorpion and Sunrise technology and also of intercalating molecules with double- stranded nucleic acid sequences such as saturating dyes.

In particular, the method to prepare a crude biological sample described here is also compatible with RNA analysis methods based on High Resolution Melting (HRM) analysis.

DESCRIPTION OF THE DRAWINGS These and other aspects, characteristics and advantages of the present invention will become apparent from the following description of some embodiments, given as a non-restrictive example with reference to the attached drawings wherein:

- fig. 1 is a graph that shows RNA amplification curves in which the curves were obtained using a biological sample prepared in accordance with the present invention (curve A), extracted and purified with known systems (curve B) and used crude (curve C).

- fig. 2 is a graph that shows HRM analysis curves of a sample prepared in accordance with the present invention (dashed line), extracted and purified with known systems (dashed line) and used crude (dotted line);

- fig. 3 is a graph that shows RNA amplification curves from which it is possible to infer the amplification efficacy achieved by combining more than one proteolytic enzyme (curve 3.1) compared to the use of a single enzyme (curves 3.2 and 3.3);

- fig. 4 is a graph that shows RNA amplification curves from which it is possible to infer the amplification efficacy both in the presence of added final preservative solution (curve 4.1) and also in the absence of final preservative solution (curve 4.2). DESCRIPTION OF SOME EMBODIMENTS

We will now refer in detail to the possible embodiments of the invention, of which one or more examples are shown in the attached drawings, by way of a nonlimiting illustration. The phraseology and terminology used here is also for the purposes of providing non-limiting examples. All measurements are carried out at 25°C and at atmospheric pressure, unless otherwise indicated. All temperatures are in degrees Celsius, unless otherwise indicated.

All percentages and ratios indicated shall be understood to refer to the weight of the total composition (w/w), unless otherwise indicated. All percentage ranges indicated here are given with the provision that the sum with respect the overall composition is 100%, unless otherwise indicated.

All the intervals reported here shall be understood to include the extremes, including those that report an interval “between” two values, unless otherwise indicated. The present description also includes the intervals that derive from uniting or overlapping two or more intervals described, unless otherwise indicated.

The present description also includes the intervals that can derive from the combination of two or more values taken at different points, unless otherwise indicated. Some embodiments of the present invention concern a method to prepare a crude biological sample to supply viral RNA suitable to be detected by means of nucleic acid amplification and hybridization techniques.

Advantageously, the crude biological sample can be saliva, sputum, vaginal mucus, bronchial mucus, nasal mucus, oropharyngeal mucus, urine, feces, whole blood, blood serum, skin secretions, spinal fluid, pulmonary and alveolar secretions.

The biological sample can be supplied by a sample performed by means of a swab, biopsy, plaster, drop in a strip of absorbent paper, needle and test tube, etc., or other similar or comparable samples.

Some embodiments of the method described here comprise:

- supplying a crude biological sample in liquid form or resuspended in an aqueous solution free of protein denaturing substances, - combining the crude sample with a lysis solution containing one or several proteolytic enzymes,

- subjecting the crude biological sample combined with the lysis solution to a heat treatment (or first heat treatment) at a temperature between 25°C and 85°C for a time between 1 and 25 minutes, - subjecting at least part of the biological sample combined with the lysis solution of the previous step to an additional heat treatment (or second or last heat treatment) at a temperature between 70°C and 250°C for a time between 3 seconds and 45 minutes.

In some embodiments, in particular, the method as above can provide to resuspend or dilute the crude biological sample in a lysis buffer containing the one or several proteolytic enzymes.

The one or several proteolytic enzymes can be natural or recombinant, for example.

The preparation method described here is favorably compatible with many known aqueous solutions for the preservation of biological samples, in particular those in which it is provided to perform viral RNA analyzes. The preparation method described here, therefore, can be used with a biological sample preserved or resuspended in water, phosphate buffer, saline solution, sample transport and preservation solutions such as UTM® or Xpert®, or other solutions that generally do not contain protein denaturing substances.

Substances that can denature or alter the structure of proteins can be acids, alkalis, heavy metal salts, ethanol, guanidine detergents, guanidine chloride, guanidinium thiocyanate, urea, formamide, guanidine, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, ammonium thiocyanate, phenol.

According to some embodiments, it can be provided to dilute the crude biological sample, before subjecting it to the first heat treatment, with an aqueous solution free of protein denaturing substances. Some embodiments can provide that the crude biological sample is supplied in dried form prior to the resuspension in the aqueous solution free of protein denaturing substances.

The preparation method can therefore be used on a crude biological sample obtained with a swab that has not been preserved in an aqueous solution, after resuspension thereof, ensuring however that RNA of a quality suitable for the detection is provided.

The method described here is therefore suitable to prepare crude biological samples left to dry accidentally or intentionally. In the latter case it is therefore possible, where appropriate, to preserve the biological sample without using preservative solutions, thus making the preservation of the biological sample itself more economical, simple and flexible.

The first treatment as above is of the thermal-enzymatic type and causes a lysis aimed at releasing the RNA from the infectious particles.

Some embodiments provide to combine the crude sample in the lysis solution to obtain a solution from 1 to 99% w/w of crude sample.

In accordance with possible embodiments, the one or several proteolytic enzymes are selected from a group consisting of: trypsin, furin, proteinase K, endoproteinase, elastase, streptogrisin, thrombin, pepsin, chymostrypsin, papain, bromelain, pancreatin, amylase, cysteine protease, aspartate protease, carboxy protease.

In accordance with possible embodiments, it is also possible to use more than one proteolytic enzyme in the lysis solution, for example two suitably selected proteolytic enzymes, or three proteolytic enzymes or even more than three. A possible implementation can provide to use, for example, trypsin and proteinase K in the lysis solution. In other implementations, one or several additional proteolytic enzymes can also be provided in the lysis solution, in addition to trypsin and proteinase K.

The proteolytic enzymes allow the digestion of the proteins present in the biological sample, they help to inactivate the infectivity of some pathogens, if present, and help to degrade the RNases, protecting the RNA for its use in the subsequent detection analysis, for example PCR.

In accordance with some embodiments, the lysis solution preferably contains proteinase K and trypsin between 0.1 pg/mL and 10 mg/mL.

The additional (or second or last) heat treatment mentioned above is performed, on the other hand, with the aim of inactivating the proteases contained in the lysis solution, which would otherwise unintentionally act on the proteins/enzymes used for subsequent analyzes, such as RT-PCR. At the end of the additional (or second or last) heat treatment, the crude biological sample presents, in solution, RNA suitable to be detected.

The crude biological sample thus prepared and treated can possibly be mixed with a preservative solution to allow its preservation preventing the degradation of the RNA. The preserving solution advantageously allows to inhibit any residual RNases which could degrade the RNA present in the biological sample and compromise subsequent analyzes, especially if the analyzes are not conducted immediately after treatment.

The preservative solution can contain protein and/or inorganic ribonuclease inhibitors.

The protein inhibitors can be selected from a group which comprises antibodies specific against ribonucleases.

The present invention is capable of supplying an RNA suitable to be used in subsequent detection methods, which make use of intercalating molecules with double-stranded nucleic acid sequences, fluorescent probes used in techniques such as RT-PCR.

In particular, the preparation method is also compatible with RNA analysis methods based on HRM analysis.

The intercalating molecules can be any sensor or reporter molecule configured to emit a signal that can be detectable by a reader that analyzes a variation of an electrical signal in terms of inductance, current, electrical potential, in the case of conductometry, amperometric or voltimetric detection, or the presence of light at specific wavelengths in case of fluorescence/chemiluminescence detection, or light deflection and/or refraction/diffraction phenomena, in case of optical plasmonic detection.

In some implementations, the intercalating molecules are fluorescence emitting intercalating molecules. According to such implementations, the fluorescent intercalating molecules can be one or several of the following molecules: SYTO- 9, SYTO-13, SYTO-16, SYTO-64, SYTO-82, YO-PRO-1, SYTO-60, SYTO-62, TOTO-3, POPO-3, BOBO-3.

The present invention can also provide a kit for preparing the crude biological sample to supply viral RNA suitable to be detected by means of nucleic acid hybridization and amplification techniques.

This kit can comprise:

- an aqueous solution not containing protein denaturing substances,

- a lysis solution containing one or several proteolytic enzymes.

In some embodiments, the kit comprises a suitable container, such as a vial, flask or test tube, containing the aqueous solution not containing protein denaturing substances and another suitable container, such as a vial, flask or test tube, containing the lysis solution containing one or several proteolytic enzymes.

In some embodiments, the one or several proteolytic enzymes can be selected from a group consisting of: trypsin, furin, proteinase K, endoproteinase, elastase, streptogrisin, thrombin, pepsin, chymostrypsin, papain, bromelain, pancreatin, amylase, cysteine protease, aspartate protease, carboxy protease.

In some embodiments, the lysis solution can contain proteinase K and trypsin, for example between 0.1 pg/mL and 10 mg/mL.

In some embodiments, the kit can comprise a preservative solution. The preservative solution contains protein and/or inorganic ribonuclease inhibitors. The protein inhibitors can be selected from a group which comprises antibodies specific against ribonucleases.

Some embodiments provide to combine the treated crude sample solution with the preservative solution so that the resulting solution comprises from 1 to 99% w/w of treated crude sample solution.

The kit described here, in some embodiments, allows to make available a buffer formed by the above aqueous solution not containing protein denaturing substances and by the above lysis solution containing one or several proteolytic enzymes. These two solutions are added to each other, for example in a test tube or similar container, preferably sterile and free of RNases and DNases enzymes, in order to form the pre-treatment buffer. Advantageously, it is provided to add equal quantities of the two solutions. In advantageous embodiments, this buffer is suitable for the pre-treatment of nasopharyngeal samples, and it allows, by means of the two heat treatments, to directly obtain a sample available for RT-PCR analysis aimed at detecting RNA biomarkers.

This pre-treatment buffer, consisting of the two solutions as above, can be added in a certain reciprocal quantity to a nasopharyngeal sample preserved in Universal Transport Medium (UTM). The sample premixed with the pre-treatment buffer is incubated at two different temperatures for specific periods of time. During this process, the nucleic acid RNAs are solubilized and finally kept protected by a lysate stabilizer. After pre-treatment, the sample is ready to be loaded into compatible PCR reactions.

The Applicant has found that the presence of more than one proteolytic enzyme in the lysis solution is particularly advantageous, and in particular at least the combination of trypsin-protease K, compared to using single proteolytic enzymes. This approach has proved to be particularly advantageous in overcoming the problem that arises when the crude sample is lysed with a heat treatment or a proteolytic enzyme, wherein some residual RNases always remain and, if there is little starting RNA and the sample taken is not analyzed immediately, the RNA is progressively degraded by the residual RNases. RNases are very efficient and resistant enzymes, often temperature is not enough to inactivate them. Therefore, the Applicant has found that using at least two proteolytic enzymes in combination in the lysis solution as above is very efficient for removing the residual RNases.

As described above, in some embodiments a preservative solution is provided which, in possible implementations, contains protein and/or inorganic ribonuclease inhibitors. In this regard, the Applicant has also found that it can be particularly advantageous to protect the sample prepared and treated with the method described here in any case, in particular to protect it completely from residual RNases, by also combining an additional RNA-preserving solution or buffer which contains an RNases inhibitor with the pre-treatment buffer consisting of the two solutions described above. For example, and in a manner that does not non-limit the scope of protection of the present invention, it is possible to use an RNases inhibitor available on the market from Merck or from New England Biolabs Inc.

The kit described here, therefore, in addition to containing the two solutions, can also include a preservative solution which contains an RNases inhibitor. In particular, the kit can include an additional suitable container, such as a vial, flask or test tube, containing the preservative solution which contains an RNases inhibitor.

EXPERIMENTAL EXAMPLE

Fig. 1 shows a graph showing, for comparison, amplification curves obtained by means of RT-PCR of RNA coming from a same biological sample but prepared and treated in three different ways, described below.

The biological sample in question is saliva and the amplified RNA is SARS- CoV-2 viral RNA. Hereafter, by RNA we mean SARS-CoV-2 viral RNA.

In particular, curve A was obtained by amplifying the RNA of the crude biological sample prepared with the method according to the embodiments described here, in one particular case, 80 pL of saliva was diluted 1 :2 in water combined with 20 pL of a lysis solution containing proteinase K and trypsin, each one in the concentration ranges described here. The 100 pL thus obtained were subjected to the first heat treatment at 56°C for 10 minutes and subsequently to the second heat treatment at 98°C for 3 minutes; finally, between 2 and 10 pL of preservative solution (Merck - #3335402001) were added.

Curve B was obtained by amplifying the RNA of the crude biological sample extracted and purified with a known system, that is, QIAamp Viral RNA Mini Kit; curve C was obtained by amplifying the RNA of the crude biological sample neither treated, nor extracted, nor purified.

From the graph it is possible to see how the treatment and preparation of the sample with the method provided by the invention allows an amplification of the RNA comparable to, if not even greater than, that obtained from RNA extracted and purified using known technologies based on solvents and columns with high affinity for nucleic acids.

Therefore, the invention allows to obtain a solution in which the RNA is of high quality and the solvent is compatible with normal amplification techniques.

The crude biological sample used directly in the RT-PCR reaction, therefore not extracted and not treated, on the contrary, does not allow the amplification of the nucleic acid, resulting in a flat amplification line (dashed line). The graph of fig. 2 shows an HRM analysis performed on the amplicons used to generate the graph in fig. 1. The fluorescence emitted by the reporter molecule was detected with a thermal increase step of 0.1 °C on a suitable detection instrument. It can be seen how the amplicon obtained from the crude biological sample prepared according to the present invention (dashed line) has a melting curve substantially comparable to the melting curve of the amplicon obtained from the crude biological sample extracted and purified with a known system (continuous line). The experiment therefore demonstrates the compatibility of the RNA obtained with the invention with subsequent HRM analyzes.

The amplification graph in fig. 3 shows the amplification efficacy achieved by combining more than one proteolytic enzyme compared to using a single enzyme.

The amplification of curve 3.1 shows how the combination of trypsin and proteinase K can guarantee a more efficient amplification of the 10,000 SARS- CoV-2 genomes dosed in saliva samples and treated following the method described in the present invention compared to the amplifications performed with trypsin only (curve 3.2) and proteinase K only (curve 3.3).

The amplification graph in fig. 4 shows the amplification efficacy both in the presence of a final preservative solution added (curve 4.1) and also in the absence of a final preservative solution added (curve 4.2). After the lysis treatment described here, by amplifying the same quantity of genomes, amplification curves were performed by means of RT-PCR of 10,000 SARS-CoV-2 genomes. Curve 4.2 shows how the absence of preservative solution causes a degradation of the viral RNA compared to curve 4.1 where the preservative solution was added.

ANALYTICAL SENSITIVITY

Analytical sensitivity, or Limit of Detection (LoD), is defined as the lowest concentration of an analyte in a sample that can be consistently detected with a stated probability (typically 95% certainty). The Applicant used a Real Time PCR detection kit for SARS-CoV-2 virus, designed for high sensitivity detection of viral RNA in samples derived from nasopharyngeal, oropharyngeal and saliva swabs, called CoronaMelt Var SARS-CoV-2 and commercially available from A. Menarini Diagnostics.

The LoD of the test with the detection kit as above coupled with the method to prepare the sample described here, which uses the pre-treatment buffer described above, was determined by adding FDA-approved SARS-CoV-2 synthetic reference material at known concentration in UTM from negative samples. The CoronaMelt Var SARS-CoV-2 test LoD is 10 copies/reaction.

ARTIFICIAL SAMPLES

The negative samples in UTM were added to 2X and 5X LoD, that is, 20 and 50 copies of viral standard RNA, and subsequently pre-treated with the preparation method and using the kit described here. The pre-treated samples were tested with the CoronaMelt Var SARS-CoV-2 test as above. 100% of the artificial samples were successfully detected.

SENSITIVITY AND CLINICAL SPECIFICITY The clinical performance of the CoronaMelt Var SARS-CoV-2 test as above, coupled with the sample preparation method and using the kit described here were evaluated on a library of 60 remaining clinical samples of nasopharyngeal UTM, comparing the same samples subjected to pre-treatment by means of the method and using the kit described here compared to RNA extraction. In one of the embodiments of the invention, clinical sensitivity was 96.77% and clinical specificity was 93.10%. The overall agreement between the extracted samples and those treated with the sample preparation method and using the kit described here was 95% (Kappa Cohen: 0.90, near perfect agreement), demonstrating the equivalence in the results between the two methods, while overcoming the disadvantages of the standard RNA extraction technique described above, in particular related to the risk of difficulty in finding reagents for the extraction of nucleic acids.

It is clear that modifications and/or additions of step may be made to the preparation method as described heretofore, without departing from the field and scope of the present invention, as defined by the claims.