WO2021180337A1

WO2021180337A1 - Molecular fingerprinting methods to detect and genotype different rna targets through reverse transcription polymerase chain reaction in a single reaction

Info

Publication number: WO2021180337A1
Application number: PCT/EP2020/057006
Authority: WO
Inventors: Rudy IPPODRINO; Maria PACHETTI; Elisabetta MAURO; Bruna MARINI
Original assignee: Ulisse Biomed S.P.A.
Priority date: 2020-03-13
Filing date: 2020-03-13
Publication date: 2021-09-16
Also published as: EP4118232A1

Abstract

Embodiments of the present disclosure relate to primers, oligonucleotides, chemical compounds and methods for a multiplex Reverse Transcription Polymerase Chain Reaction (RT-PCR) analysis able to perform the detection and genotyping of different RNA strains of the same pathogen, or different pathogens belonging to separated genus or family, and genetic variations in a single reaction.

Description

“MOLECULAR FINGERPRINTING METHODS TO DETECT AND GENOTYPE DIFFERENT RNA TARGETS THROUGH REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION IN A SINGLE REACTION”

FIELD OF THE INVENTION

Embodiments of the present disclosure relate to primers, oligonucleotides, chemical compounds and methods for a multiplex Reverse Transcription Polymerase Chain Reaction (RT-PCR) analysis able to perform the detection and genotyping of different RNA strains of the same pathogen, or different pathogens belonging to separated genus or family, and genetic variations in a single reaction. In particular, embodiments of the present disclosure relate to primers, oligonucleotides, methods and conditions to detect and characterize different strains and point mutations of pathogens responsible for diffused and or life- threatening respiratory diseases like Severe Acute Respiratory Syndrome - CoronaVirus (SARS-CoV), SARS-CoV-2 causing the COVID-19 disease, Middle East Respiratory Syndrome - CoronaVirus (MERS-CoV), and Influenza viruses, through RT-PCR using High Resolution Melting (HRM) technology in multiplex assay format.

BACKGROUND OF THE INVENTION Reverse transcription Polymerase Chain Reaction PCR (RT-PCR) uses RNA rather than DNA as starting template. In a RT-PCR amplification, first, the enzyme reverse transcriptase uses the RNA template to produce a complementary single-stranded DNA strand called complementary DNA (cDNA) in a process known as reverse transcription. Next, DNA polymerase is used to convert the single-stranded cDNA into double-stranded DNA. These DNA molecules can now be used as templates for a PCR reaction as described above. The value of RT-PCR is that it can be used to determine if a RNA species is present in a sample or to clone a cDNA sequence for a subsequent experiment.

High Resolution Melting (HRM) is an additional post-PCR analysis step that further characterizes the amplicons by studying thermal denaturation of double- stranded DNA. This occurs through the analysis of amplicon disassociation (melting) behaviour in a ramp of temperatures usually ranging from 65°C to 95°C, with a fluorescence acquisition rating of 0.1°C/sec or less. This method allows to discriminate sequence variations and features among different amplicons, and even single nucleotide polymorphisms (SNPs) can be observed.

HRM is used in diagnostics, for example in the context of genetic tests able to identify SNPs in polymorphic alleles and it has been proposed for a variety of applications including pathogen detection and genotyping.

Nowadays HRM analysis requires highly pure extracted nucleic acids: this restricts its application mostly to high-income settings where nucleic acids extraction is automatized.

Moreover, to achieve high specificity, HRM is performed in PCR reactions requiring target-dedicated primers and probe sets, that are not usually a quite affordable reagent. Nevertheless, HRM has an enormous unexplored potential for useful applications also in low-income settings, such as the characterization identification of human pathogens.

Coronaviridae is a family of RNA-based viruses that mainly affects animals. Only few members of this family (divided in different subgroups) also affect humans, such as beta-coronavirus SARS-CoV, SARS-CoV-2 and MERS-CoV, able to trigger lethal pneumonia.

Recently, a novel beta-coronavirus triggered an epidemic started in Wuhan, China. Still the modalities of the start of the outbreak are not fully understood but it appeared that this novel Coronavirus originated from a bat coronavirus. It is possible that another species (still unknown but likely to be sold at the live- animals and seafood market in Wuhan) was the transfer species between bats and humans. Indeed, bat coronaviruses can be very similar to SARS-like viruses and represent a dangerous natural source of these viruses. The novel coronavirus is now officially named SARS-CoV-2, and very recently WHO declared that it is pandemic.

The symptoms are very similar to the ones caused by the regular flu: cough, fever, sneeze, tiredness and difficulties to breath; most cases spontaneously resolve as a regular flu but a percentage of them leads to severe pneumonia.

As soon as the first viral genomes were sequenced in China, molecular diagnostic tests were developed in order to identify viral nucleic acids. CDC (Centre of Disease Control) published several guidelines for testing and FDA (Food and Drugs Administration) approved the usage of a set of primers and probes in these emergency settings, also due to the absence of alternatives. CDC indicates that biological specimens can be tested through nasopharyngeal and oropharyngeal swabs.

Since SARS-CoV-2 has a RNA-based genome, the diagnostic assays include a reverse transcription step followed by a PCR with primers able to amplify specific parts of the viral genome.

However no tests are able to distinguish between mutations that can affect disease severity and progression or provide information about viral contagiousness.

There is therefore a need to improve molecular fingerprinting methods to identify and discriminate different RNA targets through reverse-transcriptase polymerase chain reaction coupled (RT-PCR), which overcome at least one of the drawbacks in the art.

In particular, one aim of the present disclosure is to characterize several mutation hotspots, in particular associated with disease severity or contagiousness, of infectious pathogens able to cause respiratory diseases.

SUMMARY OF THE INVENTION

According to embodiments, a molecular fingerprinting method to detect and genotyping RNA targets in a sample through RT-PCR is provided. The method of the present disclosure is able to discriminate and genotype different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations in a single reaction. In one embodiment, the method includes:

- providing a one step RT-PCR reaction mixture comprising a reverse transcription and amplification buffer comprising an intercalating molecule or compound incorporated into the double-stranded amplicon and emitting a detectable signal;

- performing in one single reaction both reverse transcription and PCR amplification using said RT-PCR reaction mixture and said sample;

- performing, at the end of the PCR amplification, a High Resolution Melting (HRM) analysis on the RT-PCR reaction mixture and the sample previously subjected to RT-PCR amplification; wherein the RT-PCR reaction mixture comprises one or more reverse transcription primers for reverting one or more RNA target molecules in cDNA molecules, wherein the RT-PCR reaction mixture comprises two or more pairs of amplification primers for amplifying, in a multiplex approach, two or more cDNA molecules, wherein said amplification primers are designed in order to generate amplicons with a different melting temperature each other in order to discriminate, in the HRM analysis, each amplicon by observing the specific melting temperature of each amplicon, wherein the method further comprises monitoring, during the HRM analysis, the change in the signal emission resulting from the temperature-induced denaturation of the double-stranded amplicons into two single-stranded DNA, due to the release of the intercalating molecule or compound, wherein the method further comprises determining discrimination and genotyping of different strains of the same pathogen, different pathogens belonging to separated genus and genetic point mutation in the sample, through a reader analysing the signal variation and obtaining the result of the analysis through a graphic interface connected to said reader.

According to further embodiments, a diagnostic kit for detection and genotyping of RNA targets is provided, comprising a RT-PCR reaction mixture that can be used to perform a reverse transcription, a PCR amplification and a subsequent HRM analysis on the RT-PCR reaction mixture previously subjected to PCR. In one embodiment, the RT-PCR reaction mixture comprises one or more reverse transcription primers for reverting, in a multiplex approach, two or more target nucleic acids, two or more pairs of amplification primers for amplifying, in a multiplex approach, two or more target nucleic acids, both RNA and DNA wherein said primers are designed in order to generate amplicons with a different melting temperature in order to discriminate, in the HRM analysis, each amplicon by observing the specific melting temperature of each amplicon thus assessing if a characteristic mutation, clinically relevant, is present into the amplicon. According to still further embodiments, amplification primers are provided for performing a molecular fingerprinting method to detect and genotype RNA targets through RT-PCR, wherein the method is able to discriminate and genotype different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations in a single reaction. In one embodiment, the amplification primers are provided for amplifying in a multiplex approach two or more target nucleic acids in a RT-PCR amplification, wherein said primers are designed in order to generate amplicons with a different melting temperature each other in order to discriminate, in a HRM analysis following the PCR amplification, each amplicon by observing the specific melting temperature of each amplicon.

According to embodiments, said primers are designed to target different RNA target molecules in order to generate amplicons with a different melting temperature in order to discriminate them, in the HRM analysis; said different target molecules can belong to different pathogens that require different management and therapy, and that can have different prognosis.

Another embodiment includes primers that are designed to target different loci of the same target molecules in order to generate amplicons with a different melting temperature in order to discriminate them, in the HRM analysis, with the aim of increasing test performance in terms of sensitivity and specificity.

According to yet further embodiments, an apparatus to perform a molecular fingerprinting method for detection and genotyping of cDNA targets in a sample through RT-PCR is provided. The method is able to discriminate and genotype different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations in a single reaction. In one embodiment, the apparatus includes:

- a RT-PCR reaction mixture comprising a reverse transcription and amplification buffer comprising an intercalating molecule or compound incorporated into the double-stranded amplicon and emitting a detectable signal;

- a PCR amplification device configured for using said PCR reaction mixture and said sample;

- a device for performing a HRM analysis on the PCR reaction mixture and the sample previously subjected to PCR amplification; wherein the RT-PCR reaction mixture comprises one or more reverse transcription primers for reverting, in a multiplex approach, two or more target nucleic acids, wherein the PCR reaction mixture comprises two or more pairs of amplification primers for amplifying in a multiplex approach two or more target nucleic acids, wherein said amplification primers are designed in order to generate amplicons with a different melting temperature each other in order to discriminate, in the HRM analysis, each amplicon by observing the specific melting temperature of each amplicon;

- monitoring means for monitoring, during the HRM analysis, the change in the signal emission resulting from the temperature-induced denaturation of the double-stranded amplicons into two single-stranded DNA, due to the release of the intercalating molecule or compound,

- a reader analysing the signal variation for determining discrimination and genotyping of different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations in the sample, so that the result of the analysis can be obtained through a graphic interface connected to said reader.

According to embodiments, combinable with all embodiments described herein, the RNA target that can be detected and genotyped is a pathogen RNA target.

Advantageously, the proposed technique exploits common cDNA intercalating molecules or compounds, such as for instance intercalating dyes, that are much more affordable than fluorescently-labelled probes.

Moreover, according to the present disclosure, the post-PCR HRM analysis does not require a dedicated instrument but it can also be performed in any thermocycler with a HRM resolution of at least 0.1°C/sec or less. In this context primer design is crucial and the primers designed according to the present disclosure are found to be fully successful in ensuring the highest specificity for each single target, given the multiplex assay format.

In advantageous embodiments, the melting fingerprinting technology according to the present disclosure has been applied to successfully improve the detection of RNA viruses triggering severe respiratory syndromes, such as coronaviruses and influenza viruses. In particular, an embodiment is able to detect SARS-CoV-2 strains, causing the COVID-19 disease; an embodiment describes a version of the assay that is able to discriminate SARS-CoV2 from other viruses causing similar symptoms but requiring different management: SARS-CoV, MERS-CoV, Influenza virus A, B, C and D. This might significantly contribute to prevent health facilities overload.

Advantageously, embodiments described herein may use specific buffer and condition, specific RNA or cDNA primers and innovative hybrid primers.

Embodiments described herein allow to discriminate, for example, SARS- CoV-2 strains through a specific HRM analysis.

Advantageously, embodiments described herein allow to detect up to 20 SARS-CoV-2 variants at the same time. The viral genome amplification reaction can be coupled to the amplification of a DNA loading control target (human RNase P gene for example).

Other pathogens that can be detect according to the present disclosure include for instance other infectious pathogens than SARS-CoV-2 responsible for epidemic and pandemic diseases, causing mainly severe respiratory syndromes, such as SARS-CoV, MERS-CoV, Influenza virus type A, B, C, D, and other coronaviruses and influenza viruses.

Embodiments described herein also provide a melting calibrator that is required to set each RT-PCR machine, for example a real-time PCR machine, for a correct and precise melting analysis required to distinguish the pathogens.

Embodiments described herein according to the present disclosure fully solve the above-mentioned issues of the tests and methods of the prior art, and further provide at least the following advantages:

- novel sample type can be used: embodiments of the present disclosure can work using RNA extracted directly from different sample types as nasopharyngeal swabs, oropharyngeal swabs, buccal swabs, sputum, saliva, nasal swabs, samples that the patients can easily self-collect with no invasiveness and no pain;

- affordability: embodiments of the present disclosure are much affordable compared to the afore-mentioned tests and methods of the prior art, because they do not use dozens of expensive labelled-probes but a unique intercalating molecule or compound, e.g. an intercalating dye can be provided; moreover the genotyping does not require incubation and reverse blot steps but it can be performed in a single short RT-PCR one-step reaction (for instance in less than 90 minutes);

- open-accessibility: embodiments of the present disclosure can be executed by any real-time PCR machine and do not require a dedicated and specific instrument;

- single-channel: result of embodiments of the present disclosure can be provided through the analysis of a single signal, e.g. fluorescence, channel, in contrast to the afore-mentioned tests and methods of the prior art, where more than one channel is used. The selected channel can be chosen among those embedded in any real-time PCR machine and this makes embodiments of the present disclosure suitable for any real-time PCR machines.

- mutation detection: RNA viruses are characterized by a higher mutation frequency rate, that renders this class of viruses particularly dangerous for public health. Indeed, given the genome instability it is very crucial to monitor genome variation in the population, in order to develop proper diagnostic strategies, therapeutics and vaccines; the embodiments allows an economically affordable and informative mutations diagnostic monitoring.

Moreover, the embodiments allow in a single reaction without requiring other dedicated machines or sequencing services to detect mutations that can be associated with increased or decreased disease severity or contagiousness, thus providing information for patients management.

These and other features, aspects and advantages of the present disclosure will become better understood with reference to the following description, the drawings and appended claims. The drawings, which are incorporated in and constitute a part of this specification, are used to illustrate embodiments of the present subject matter and, together with the description, serve to explain the principles of the disclosure.

The various aspects and features described in the present disclosure can be applied, individually, wherever possible. These individual aspects, for instance the aspects and features described in the attached dependent claims, can be made subject of divisional patent applications.

It is noted that anything found to be already known during the patenting process is understood not to be claimed and to be the subject of a disclaimer.

BRIEF DESCRIPTION OF THE DRAWINGS So that the manner in which the above recited features of the present invention can be understood in detail, a more particular description of the invention, briefly summarized above, may be had by reference to embodiments. The accompanying drawings relate to embodiments of the disclosure and are described in the following:

- Figure 1 is a graph showing Derivative Fluorescence (-d/dT) vs. Melting Temperature (°C) curves with specific primers designed to obtain different melting temperature for two SARS CoV-2 types compared with wild-type non mutated SARS CoV-2 genome, allowing simultaneous precise identification of two frequent SARS CoV-2 genome variants; an example is reported of SARS- CoV2 HRM analysis after RT-PCR of amplicon containing viral mutation, position 14408 in the viral genome. Data show results of high-resolution-melting analysis, after reverse transcriptase polymerase chain reaction, using some embodiments of the RT-PCR master mix composition described in this patent. The amplicon is totally 142 base pairs and the presence of the characterized mutation lead to a clear and detectable DNA melting shift of the wild type amplicon (grey line) versus the amplicon containing the mutation (black line) of 0,3 °C thus allowing to identify the presence of the mutation.

DETAILED DESCRIPTION OF THE EMBODIMENTS Reference will now be made in detail to the various embodiments of the invention, using the attached figures. Generally, only the differences with respect to individual embodiments are described. Each example is provided by way of explanation of the invention and is not meant as a limitation of the invention. For example, features illustrated or described as part of one embodiment can be used on or in conjunction with other embodiments to yield yet a further embodiment. It is intended that the present invention includes such modifications and variations. Before describing these embodiments, it shall be also clarified that the present description is not limited in its application to details of the construction and disposition of the components as described in the following description using the attached drawings. The present description can provide other embodiments and can be obtained or executed in various other ways. It shall also be clarified that the phraseology and terminology used here is for the purposes of description only, and cannot be considered as limitative.

All the percentages and ratios indicated refer to the weight of the total composition (for example indicated as % w/w), unless otherwise indicated. All the measurements are made, unless otherwise indicated, at 25°C and atmospheric pressure. All the temperatures, unless otherwise indicated, are expressed in degrees Centigrade.

All the ranges reported here shall be understood to include the extremes, including those that report an interval “between” two values. Furthermore, all the ranges reported here shall be understood to include and describe the punctual values included therein, and also all the sub-intervals. Moreover, all the ranges are intended as such that the sum of the values included therein, in the final composition, gives 100%, in particular considering that the person of skill will know how to choose the values of the ranges so that the sum does not exceed 100%.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. In addition, it will be readily apparent to one of ordinary skill in the art in light of the teachings herein that certain changes and modifications may be made thereto without departing from the spirit and scope of the appended claims. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. To the extent such publications may set out definitions of a term that conflicts with the explicit or implicit definition of the present disclosure, the definition of the present disclosure controls. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Embodiments of the present disclosure generally relate to molecular fingerprinting method to detect and characterize RNA targets in a sample through RT-PCR is provided.

The method of the present disclosure is able to discriminate and genotype different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations in a single reaction. In one embodiment, the method includes:

- providing an one step RT-PCR reaction mixture comprising a reverse transcription and amplification buffer comprising an intercalating molecule or compound incorporated into the double-stranded amplicon and emitting a detectable signal;

- performing, at the end of the RT-PCR amplification, a HRM analysis on said RT-PCR reaction mixture and said sample previously subjected to RT-PCR amplification; wherein the RT-PCR reaction mixture comprises one or more reverse transcription primers for reverting one or more RNA target molecules into cDNA molecules, wherein the RT-PCR reaction mixture comprises two or more pairs of amplification primers for amplifying in a multiplex approach two or more cDNA molecules, wherein said amplification primers are designed in order to generate amplicons with a different melting temperature each other in order to discriminate, in the HRM analysis, each amplicon by observing the specific melting temperature of each amplicon, wherein the method further comprises monitoring, during the HRM analysis, the change in the signal emission resulting from the temperature- induced denaturation of the double-stranded amplicons into two single-stranded cDNA, due to the release of the intercalating molecule or compound, wherein the method further comprises determining discrimination and genotyping of different strains or mutations of the same pathogen, different pathogens belonging to separated genus and genetic variations in the sample, through a reader analysing the signal variation and obtaining the result of the analysis through a graphic interface connected to said reader.

According to embodiments, the pathogen is an infectious pathogen able to cause a respiratory diseases, in particular, the pathogen is SARS-CoV2. In preferred embodiments, the amplification primers include one or more specific primers for different loci of the pathogen that, preferably, contain “hotspot site” for mutation(s).

In particular, an embodiment is able to detect and genotype SARS-CoV2 strains RNA.

According to embodiments, the amplification specific primers are able to amplify and identify, through HRM analysis, amplicons containing the following SARS CoV-2 genome mutation positions: 155, 883, 1189, 1397, 3036, 8782 (lit.), 9438, 11083, 14408, 21767, 23403, 25320, 25653, 26143, 27045, 28144 (lit.), 28688, 28881, 29095

In some embodiments detection and genotyping of viral mutations can be used to design therapeutic small molecules capable to defeat SARS CoV-2 infection or decrease viral infectivity and pathogenicity through the interaction of those molecules with specific protein epitopes whose shape is influenced and modulate by the presence one or more of the said RNA SARS CoV-2 genome mutations.

In some embodiments detection and genotyping of viral mutations can be used to design specific biological drugs capable to defeat SARS CoV-2 infection or decrease viral infectivity and pathogenicity through the interaction of those molecules with specific protein epitopes whose shape is influenced and modulate by the presence one or more of the said RNA SARS CoV-2 genome mutations.

In some embodiments detection and genotyping of viral mutations can be used to design specific therapeutic small RNA, short Hairpin RNA, microRNA, siRNAs capable to target RNA viral genome of SARS CoV-2 triggering its genome degradation.

In some embodiments detection and genotyping of viral mutations can be used to design vaccines, capable to prevent or defeat SARS CoV-2 infection through the interaction of antibodies, raised consequently patient vaccination, with specific protein epitopes whose shape is influenced and modulate by the presence one or more of the said RNA SARS CoV-2 genome mutations.

Advantageously, using the embodiments described herein, it is possible to detect and discriminate: - different strains and variants of a specific pathogen, each of them characterized by a mutation that can affect, for instance, disease severity or viral contagiousness (e.g. different mutants of SARS-CoV-2);

- different loci of a specific pathogen (e.g. different loci of the same variant SARS-CoV-2), in order to increase with double/triple check test performance in terms of specificity and sensitivity;

- different pathogens (e.g. different infectious pathogens responsible for severe respiratory syndromes able to turn into epidemic or pandemic, such SARS-CoV, MERS-CoV, Influenza virus A, B, C, D, or different novel coronaviruses and influenza viruses);

- different transcripts of a pathogen (e.g. Human Papillomavirus E6/E7 mRNA production as example for a DNA target pathogen; Influenza virus RNA as example for a RNA target pathogen);

Advantageously, the sample can be a crude sample. The crude sample can be nasopharyngeal swabs, oropharyngeal swabs, buccal swabs, sputum, saliva, nasal swabs, vaginal or cervical mucus, other bodily fluids, blood, urine, biopsies, formalin-fixed paraffin-embedded ( FFPE ) tissue, cells, fine needle aspiration biopsies or similar. The crude sample can be diluted prior to performing the RT- PCR amplification and HRM analysis.

According to possible embodiments, combinable with all embodiments described herein, the signal variation between an input and an output signal can be detected in a circuit included in the reader, wherein said variation is a function of the presence, amount, genotype of different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations present in the sample.

According to embodiments, combinable with all embodiments described herein, performing PCR amplification using said PCR reaction mixture includes amplifying the target purified nucleic acid using said PCR reaction mixture to generate an amplicon or amplification product.

According to embodiments, combinable with all embodiments described herein, the amplification primers are sufficiently complementary to the target nucleic acid to hybridize therewith and trigger polymerase-mediated synthesis. In one specific embodiment, combinable with all embodiments described herein, the amplification primers are designed to amplify specifically pathogen RNA (e.g. SARS-CoV-2 strains) reverted cDNA targets, producing corresponding amplicons, each from 50 to 300 base pairs (bps).

In one further specific embodiment, combinable with all embodiments described herein, the amplification primers are designed to amplify an amplicon wherein the melting peak of the amplicon is between 65°C and 95°C.

In some embodiments, the amplification primers used in RT-PCR reaction contain at 3 ’-OH primer end sequence complementary to the RNA mutation of interest which allows, in stringent conditions, to amplify only the respective cDNA amplicon which contains the mutation of interest. Specific mutated sequences are selectively amplified even in samples where the majority of the sequences do not carry the mutation. When the amplification primer is fully matched to cDNA template the amplification proceeds with full efficiency. When the 3' base is mismatched, only low-level background amplification occurs. This strategy it is based on the principle that amplification is efficient when the 3' terminal base of the primer matches the template, whereas amplification is inefficient or even nonexistent when there is a mismatch.

In still another embodiment, combinable with all embodiments described herein, each amplification primer is present in the RT-PCR reaction mixture at a final concentration range from 50 to 1000 nanomolar (nM).

According to embodiments, combinable with all embodiments described herein, the RT-PCR reaction mixture comprises the reverse transcription primers, the amplification primers and the reverse transcription and amplification buffer.

According to embodiments, the reverse transcription and amplification buffer is comprised in a diagnostic kit that is part of the present disclosure.

In one embodiment, combinable with all embodiments described herein, besides the above mentioned one or more reverse transcription primers and two or more pairs of amplification primers, the RT-PCR reaction mixture comprises a Reverse Transcriptase and a DNA polymerase. The Reverse Transcriptase and the DNA polymerase are included in said reverse transcription and amplification buffer. The Reverse Transcriptase is able to copy RNA strands into a DNA strand. The DNA polymerase is an enzyme that polymerizes new DNA strands. For instance, heat resistant or heat stable polymerase can be used, since it is more likely to remain intact during the high-temperature DNA denaturation process. One example of heat resistant or heat stable polymerase that can be used in embodiments described herein is taq polymerase. Moreover, the polymerase that can be used in association with embodiments described herein is a hot-start polymerase. In a possible implementation, the hot-start polymerase can be (Hot Start) @Taq DNA Euroclone, (Hot Start) Phire Thermo Scientific, (Hot Start) Phusion Thermo Scientific, or (Hot Start) Gold Taq polymerase Sigma. In a possible implementation and embodiment, the reverse transcriptase used can be Superscript IV One-step RT-PCR (Thermo Fischer Scientific), TaqPath™ 1-Step Multiplex Master Mix, Power SYBR® Green RNA-to-CT™ 1-Step, EXPRESS One-Step SYBR™ GreenER™, Maxima H Minus cDNA Synthesis Master Mix.

In one further embodiment, combinable with all embodiments described herein, the RT-PCR reaction mixture may further include deoxynucleoside triphosphates (dNTPs) or analogues. dNTPs or analogues are included in said amplification buffer. dNTPs or analogues are used to provide the building blocks from which the polymerase synthesize a new DNA strand. dNTPs can be substituted by functional analogues like adenine, cytosine, guanine, thymine, uracil, orotidine, inositate, xanthylate.

As above described, the RT-PCR reaction mixture comprises said intercalating molecule or compound, being incorporated into the double-stranded amplicon or amplification product and emitting fluorescence or any other detectable signal. The intercalating molecule or compound can be included in said reverse transcription and amplification buffer.

In particular, according to embodiments, the intercalating molecule can be any sensor or reporter molecule emitting a signal that can be detected by a reader analysing an electric signal variation in terms of inductance, current, electric potential, in case of conductometric, amperometric, voltammetric detection, or the presence of light at specific wavelengths, in case of a fluorescence/chemiluminescence detection, or light scattering and/or refraction/diffraction phenomena, in case of a plasmonic optical detection.

For instance, in some implementations the intercalating molecule or compound can be an intercalating dye emitting fluorescence. According to possible implementations, specific DNA intercalating dye, at a final concentration range from 0,4 to 9 mM, can be one or more of the following dyes: SYTO-9, SYTO-13, SYTO-16, SYTO-64, SYTO-82, YO-PRO-1, SYTO-60, SYTO-62, TOTO-3, POPO-3, BOBO-3, doxorubicin-conjugated quantum dot nanoparticles or similar.

In yet another embodiment, combinable with all embodiments described herein, the PCR reaction mixture may further comprise a buffer solution. The buffer solution is included in said reverse transcription and amplification buffer. The buffer solution provides a suitable chemical environment for optimum activity and stability of DNA polymerase and Reverse Transcriptase. For instance, the buffer solution may comprise water, in particular deionized water, TrisHCl and/or KC1 and possibly in some cases MgCl₂.

In one embodiment, combinable with all embodiments described herein, the RT-PCR reaction mixture may further comprise a pH stabilizer.

In one further embodiment, combinable with all embodiments described herein, the RT-PCR reaction mixture may further comprise preservatives.

In still another embodiment, combinable with all embodiments described herein, the RT-PCR reaction mixture may further comprise water.

In yet another embodiment, combinable with all embodiments described herein, the RT-PCR reaction mixture may further comprise a source of monovalent or bivalent cations. The source of monovalent or bivalent cations is composed of said amplification buffer. For example, a chloride containing monovalent ion or bivalent ions can be used. As a source of monovalent cations, potassium ions can be used. K⁺ can be obtained from potassium salts, e.g. potassium chloride, in particular potassium chloride at a concentration of about 0.1 M. As a source of bivalent cations magnesium or manganese ions can be used. Mg can be obtained from magnesium salts, e.g. magnesium chloride.

In one further embodiment, combinable with all embodiments described herein, the RT-PCR reaction mixture may further comprise bovine serum albumin (BSA). The BSA is included in said reverse transcription and amplification buffer.

In one further embodiment, combinable with all embodiments described herein, the RT-PCR reaction mixture further comprises one or more detergents. In possible implementations, said detergent can be Nonidet-P40 (NP40) at a concentration from 0.1 to 1%.

In still another embodiment, combinable with all embodiments described herein, the RT-PCR reaction mixture may further comprise additives. The additives can be included in some embodiments of the above-mentioned amplification buffer. In possible implementations, the additives that can be used are selected among one, more or all of additives in a group comprising: NP40, DMSO, TMAC (Tetramethylammonium Cloride), Acetamide, Triton, Formamide, Betaine, E. Coli ssDNA binding protein, Glycerol, L-Camitine and Gelatine. Advantageously, in embodiments exploiting a fluorescence detection, the presence of additives can be important to avoid a high basal fluorescence background allowing an increased diagnostic sensitivity, specificity and accuracy. In some possible implementations, the additives may further comprise a gelatin, for example at a concentration of about 0.1%. In yet further possible implementations, the additives may further comprise an enhancer. For example, the enhancer can be L-Camitin at a concentration of about 0,42 M. In still further possible implementations, the additives may further comprise sugar alcohol, for example sorbitol at a concentration of about 25mM.

According to embodiments, combinable with all embodiments described herein, amplifying the target purified nucleic acid using said RT-PCR reaction mixture, to generate an amplicon or amplification product, includes thermocycling by performing a ramp of temperature steps. In one possible implementation, a ramp of temperature includes performing the following temperature steps: reverse transcription at 40-65°C from 1 to 50 minutes denaturation at 95-98°C from 1 to 30 seconds; annealing in a range between 45 °C and 70°C from 1 to 60 seconds; extension for DNA polymerase with a range between 60°C and 75°C from 0 second to 5 minutes.

In possible implementations, the number of cycles of thermocycling is of at least 30 cycles, for instance between 30 and 50 cycles. One possible example is 35 cycles. In one possible implementation, hot-start polymerase can be used. Hot-start PCR avoids a non-specific amplification of DNA by inactivating the polymerase at lower temperatures, for instance through antibodies interaction, chemical modification or aptamer technology. Typically, a specific inhibitor, such as an aptamer-based inhibitor or specific antibodies can be used to block the polymerase at lower temperatures. If hot-start polymerase is used, an initial incubation step which ranges from 95°C to 98°C for 1 second to 10 minutes is performed. This initial incubation step is necessary for activation of polymerase.

According to embodiments, combinable with all embodiments described herein, the method includes, during thermocycling in the RT-PCR amplification, performing the monitoring emission signal changing, e.g. fluorescence, resulting from the temperature-induced denaturation of the double-stranded amplicons or amplification products into two single-stranded DNA, due to the releasing of the intercalating molecule or compound, i.e. intercalating dye. The intercalating molecule or compound binds to DNA in the double-strand configuration. At each amplification cycle, amplicons are generated in which the intercalating molecule or compound binds in the extension step, during which the signal (e.g. fluorescence) is acquired.

Advantageously, the RT-PCR reaction can occur in a real-time PCR machine, that allows monitoring the change in the signal, e.g. fluorescence, emission at each amplification cycle in the RT-PCR amplification, in turn allowing quantification of the presence of amplicons and quantification, therefore, of the target DNA in the amplification phase.

In other embodiments, the RT-PCR amplification may occur in a thermocycling machine able to acquire said signal emission each 0.1°C/second or less.

According to embodiments, combinable with all embodiments described herein, the HRM analysis includes performing a ramp of temperature on the RT- PCR reaction mixture previously subjected to RT-PCR amplification. In one possible implementation, a ramp of temperature includes performing the following temperature step:

• ramping from 65°C up to 95°C and from 95°C to 65°C variating the temperature 0.1°C/second or less, and performing said monitoring the change in the signal, e.g. fluorescence, emission resulting from the temperature-induced denaturation of the double-stranded amplicons or amplification products into two single-stranded DNA, due to the release of the intercalating molecule or compound, i.e. intercalating dye.

Advantageously, monitoring the change in the signal, e.g. fluorescence, emission in the HRM analysis, during which the large quantity of viral amplicons generated after the plurality of amplification thermocycling, allows to analyse the melting features of such amplicons at different temperatures. At low temperature, e.g. 60°C, amplicons are all double-stranded and the maximum level of signal, e.g. fluorescence, is detected. By slowly increasing temperature, however, amplicons start to denaturate up to complete separation into two single-stranded DNA and at this point the signal, e.g. fluorescence, will not be generated anymore. For instance, the shape and development of the Derivative Fluorescence vs. Temperature curves, as shown in Figure 1, that can be generated by the above-mentioned monitoring, change depending on the sequence. The present disclosure exploits this feature to discriminate the viral genotype or identify specific viral mutation, because the primes used are designed such as to generate amplicons with a specific and different melting temperature each. In other words, the amplification primers designed according to the present disclosure allow to amplify amplicons with a precise and specific melting temperature fingerprint each.

Therefore, according to advantageous embodiments using a real-time PCR machine, after monitoring the change in in the signal, e.g. fluorescence, emission at each amplification cycle in the real-time PCR, it will be possible to know if the analysed sample is infected or not by a pathogen or a group of pathogens, e.g. a virus, and also by which one of the possible genotypes that can be detected; the presence of different mutations, affecting differently viral pathogenesis, patients’ management and treatment, can be distinguished.

Moreover, according to the present disclosure, after monitoring the change in the signal, e.g. fluorescence, emission in the HRM analysis it will be possible to know, in the case that a sample is infected, also exactly the specific genotype or strain of the pathogen infecting the sample, or which exact pathogen of the group of pathogens. The overall result is therefore that it will be possible, in advantageous embodiments using the real-time PCR, to know, via the real-time PCR, if a patient if positive or not to a specific pathogen, or a group of pathogens and, when positive, to know, via HRM analysis, the genotype or strain of pathogen that is infecting the patient or to know the specific pathogen from the detected group of pathogens. It is possible to detect specific mutations, for instance but not only point mutations, that are associated with different disease severity status or viral contagiousness.

According to embodiments, combinable with all embodiments described herein, RT-PCR amplification can be for instance performed in a PCR thermocycler.

According to further embodiments, combinable with all embodiments described herein, RT-PCR amplification can be typically performed in a real-time PCR machine, for instance a real-time PCR thermocycler.

Since, according to the present disclosure, the same sample is first subjected to the RT-PCR amplification and then to HRM analysis, the whole method can be performed in a single apparatus, in particular a real-time PCR machine.

According to possible embodiments, the RT-PCR amplification and detection can be performed simultaneously by means of Real Time PCR in any setup known in the art, including quantitative Real time PCR allowing assessment of the pathogenic load in the infected sample, followed by HRM analysis, performed in the same real-time PCR machine.

However, in other embodiments, the two operations, i.e. RT-PCR amplification and HRM analysis, can also be performed in separate and distinct apparatuses coupled or associated each other, for instance a typical thermocycler for the RT-PCR amplification and then a real-time PCR configured for HRM analysis.

For example, in possible implementations, the detection can be performed using a dedicated PCR device, also in portable format, containing a specific Peltier module coupled with a fluorescence optical reader or other appropriate reading device, able to perform HRM analysis.

In still further implementations, the detection via the RT-PCR amplification can be performed using a dedicated PCR device containing for instance a specific Peltier module coupled to a read-out device different than a fluorescent read-out device, for instance a chemiluminescent or electrochemical read-out device, a conductimetric, amperometric, voltammetric read-out device, plasmonic optical red-out device or any other suitable read-out device.

Embodiments described herein can be used for diagnostic purposes. In particular, in the following, specific ranges of the reagents present in the two possible implementations of the PCR reaction mixture are described, that can be used for diagnostic purposes. Subsequently, specific ranges are described that can be used for specific detection of SARS-CoV-2.

In particular, the method and diagnostic kit containing the above-mentioned PCR reaction mixture according to the present disclosure can be used to detect clinically relevant pathogens present in the sample, including providing qualitative information about the presence of a mutation hotspot.

In one embodiment, a possible first amplification buffer for diagnostic purposes comprises the dNTPs, the source of mono or divalent cations, the buffer solution, the BSA, the DNA polymerase, the reverse transcriptase, the intercalating molecule or compound. For instance, one specific implementation of the first amplification buffer comprises: a) dNTPs (final concentration range: from 0.03 mM to 0.4mM) b) MgCl2 (final concentration range: from 0.15 mM to 6 mM) c) TrisHCl buffer solution (final concentration range: from 5 mM to 75 mM; pH from 4.50 to 11.00) d) KC1 (final concentration range: from 5 mM to 75 mM) e) BSA (final concentration range: from 0.001 to 0.08 mg/ml) f) DNA polymerase g) Reverse Transcriptase h) SYTO-9 (final concentration range: from 0.5mM to 9mM).

In another possible embodiment, a possible alternative second amplification buffer for diagnostic purposes is provided, that comprises the dNTPs, the source of mono or bivalent cations, the buffer solution, BSA, the DNA polymerase, the reverse transcriptase, the intercalating molecule or compound and the above mentioned additives. By the addition of additives, the second amplification buffer can be used as a PCR enhancer buffer providing increased diagnostic sensitivity, specificity and accuracy as above discussed. For instance, one specific implementation of the alternative second amplification buffer comprises: a) dNTPs (final concentration range: from 0.03 mM to 0.4mM) b) MgCl (final concentration range: from 0.15 mM to 6 mM) c) TrisFICl buffer solution (final concentration range: from 5 mM to 75 mM; pH from 4.50 to 11.00) d) KC1 (final concentration range: from 5 mM to 75 mM) e) BSA (final concentration range: from 0.001 to 0.08 mg/ml) f) DNA polymerase g) Reverse Transcriptase h) SYTO-9 (final concentration range: from 0.5mM to 9mM). i) MAC (Tetramethylammonium Chloride) (final concentration range: from 5mM to 200 mM) j) Acetamide (final concentration range: from 0% to 10%) k) Formamide (final concentration range: from 0% to 10%) l) Betaine (final concentration range: from 0 mM to 8 mM) m) Gelatine (final concentration range: from 0 mg/ml to 3.5 mg/ml).

In the embodiments of methods and diagnostic kit described herein for diagnostic purposes, since different PCR machine, i.e. different thermocycler models, may generate different curves and in particular different melting peaks, a calibrator is provided to set each real-time PCR machine for a correct and precise melting analysis required to genotype the pathogen. Indeed, some variations might occur due to machine type, efficiency due to maintenance status or acquisition settings, and the calibrator allows the adjustment of the observed measurements in a specific machine. The calibrator can be composed by synthetic oligonucleotides corresponding to the amplicons generated by the specific primers of the RT-PCR reaction mixture according to the present disclosure. Advantageously, a machine-specific calibrator can be loaded in PCR runs periodically to check the effective melting temperature of the amplicons of a particular thermocycler machine, and compare it with the expected melting temperature.

Further embodiments described herein for diagnostic purposes provide primers for obtaining combinations of melting temperature, in order to increase the number of targets detectable in the same assays. The limit of the number of targets simultaneously detectable in a single well of a PCR machine is generally defined by the capability of the system to distinguish and resolve two proximal peaks. However, according to embodiments described herein, it is possible to increase the number of targets detectable in the same assays, providing two or more sets of primers that are specific for the same cDNA targets. According to embodiments, a first set of primers is present in a RT-PCR reaction mixture in one well, at least a second set is present in another well. Each set of primers comprises primers each recognizing one specific cDNA target. The cDNA targets recognized by the first set of primers are the same as the cDNA targets recognized by the second set of primers and the melting temperature of an amplicon generated by a primer of one set of primers recognizing a specific cDNA target is different from the melting temperature of an amplicon generated by a primer of the other set of primers recognizing said specific cDNA target.

This variant is particularly useful when the application aims at differentiating high number different target.

In the following, embodiments of methods and diagnostic kit according to the present disclosure are described for specific use for SARS-CoV-2 diagnosis, using specific primers.

In one embodiment, methods and diagnostic kit of the present disclosure are used to detect SARS-CoV-2 RNA in clinical samples and to discriminate different SARS-CoV2 strains, each of them characterized by a mutation pattern profile, by HRM analysis.

According to one embodiment, the methods and diagnostic kit, in addition to any of the afore-described combination of reagents required for carrying out reverse transcription coupled in one step with amplification by PCR, includes specific primers for different viruses, different mutants of the same viruses, or different loci of the same target. The afore-mentioned specific amplification primers for SARS-CoV-2 diagnosis are characterized by the following features:

1) the difference in melting temperature of different primers does not exceed 30°C.

2) amplify pathogen RNA reverted cDNA targets producing corresponding amplicons, each from 38 to 1500 bps. 3) amplify an amplicon wherein the melting peak of the amplicon is between 65°C and 95°C.

4) each amplification primer is present in the RT-PCR reaction mixture at a final concentration range from 50 to 1000 nM.

In yet further embodiments, one further set of normalizing primers can be provided, for the reverse transcription and amplification of human RNA, said amplification of human RNA serving as an internal PCR validation control and/or control for normalization of the amplified pathogen RNA obtained, e.g. SARS-CoV-2 obtained according to any of the embodiments described herein. An example of such pair of normalizing primers targeting a fragment is targeting the human RNase P transcript.

In a preferred embodiment of the diagnostic kit for SARS-CoV-2 diagnosis, specific primers for the most conserved loci of the viral genome are provided in the RT-PCR reaction mixture of the present disclosure. As well, specific primers generating amplicons, that can be distinguished through the invention here described, containing specific mutations (e.g. point mutations) of clinical relevance because associated to a differential risk for disease severity of viral contagiousness.

In another embodiment, a diagnostic kit is able to detect and distinguish SARS-CoV-2, SARS-CoV, MERS-CoV, and Influenza viruses (e.g. A, B, C, and D)

In one embodiment, a specific reverse transcription and amplification buffer for SARS-CoV2 diagnosis comprises the following reagents with concentration expressed as ranges: a) dNTPs (final concentration range: from 0.03 mM to 0.4mM) b) MgCl₂ (final concentration range: from 0.15 mM to 6 mM) c) TrisHCl buffer solution (final concentration range: from 5 mM to 75 mM; pH from 4.50 to 11.00) d) KC1 (final concentration range: from 5 mM to 75 mM) e) BSA (final concentration range: from 0.001 to 0.08 mg/ml) f) DNA polymerase g) Reverse Transcriptase h) SYTO-9 (final concentration range: from 0.5mM to 9mM). One example of possible specific concentration values of the reagents is the following: a) dNTPs (final concentration 0.2mM) b) MgCl₂ (final concentration 0.75mM) c) TrisHCl buffer solution (final concentration 30mM: and pH 9.0) d) KC1 (final concentration 50mM) e) BSA (final concentration lOpg/ml) f) DNA polymerase g) Reverse Transcriptase h )SYTO-9 (final concentration 4mM).

In another possible embodiment, a further alternative possible specific amplification buffer for SARS-CoV-2 diagnosis, providing increased diagnostic sensitivity, specificity and accuracy comprises the following reagents with concentration expressed as ranges: a) dNTPs (final concentration range: from 0.03 mM to 0.4mM) b) MgCl (final concentration range: from 0.15 mM to 6 mM) c) TrisHCl buffer solution (final concentration range: from 5 mM to 75 mM; pH from 4.50 to 11.00) d) KC1 (final concentration range: from 5 mM to 75 mM) e) BSA (final concentration range: from 0.001 to 0.08 mg/ml) f) DNA polymerase g) Reverse Transcriptase h) SYTO-9 (final concentration range: from 0.5mM to 9mM). i) TMAC (Tetramethylammonium Cloride) (final concentration range: from 5mM to 200 mM) j) Acetamide (final concentration range: from 0% to 10%) k) Formamide (final concentration range: from 0% to 10%) l) Betaine (final concentration range: from 0 mM to 8 mM) m) Gelatine (final concentration range: from 0 mg/ml to 3.5 mg/ml).

One example of possible specific concentration values of the reagents of this further alternative amplification buffer is the following: a) dNTPs (final concentration 0.15 mM) b) MgCl₂ (final concentration 0.75 mM) c) TrisHCl buffer solution (final concentration 30mM; pH 9) d) KC1 (final concentration: 40mM) e) BSA (final concentration 10pg/ml) f) DNA polymerase g) reverse transcriptase h) SYTO-9 (final concentration 2 mM) i) TMAC (Tetramethylammonium Chloride, final concentration of 75 mM) j) Acetamide (final concentration 3%) k) Formamide (final concentration 1,5%) l) Betaine (final concentration 0,5M) m) Gelatine (final concentration 0,lmg/mL).

Advantageously, the diagnostic SARS-CoV-2 test based on the embodiments described herein can provide important information about SARS-CoV-2 spread and prognosis:

1) a diagnostic information: the amplification curves obtained by the PCR allow the detection of one or more loci of SARS-CoV-2 and thus, a sample is positive when the amplification curve occurs before 35 PCR cycles using a fluorescence threshold that range from 250.000 to 400.000.

2) the melting fingerprinting analysis, obtained by the HRM analysis, when used to the embodiments able to distinguish SARS-CoV-2 from SARS-CoV, MERS-CoV, Influenza viruses (A, B, C, D) allows the discrimination of all the above-mentioned pathogens that shows initial similar symptoms. Indeed nowadays cold, flu, throat ache and pneumonia are symptoms shared by pathologies of different severity. Considering the pandemic declared in 2020 by WHO about SARS-CoV-2 and COVID-19 disease, it is needed a tool to assess, distinguish and manage regular cold and seasonal flu cases, differentiating them immediately from diseases caused by SARS-CoV-2 and similar pathogens thus preventing health facilities overload.

3) the melting fingerprinting analysis, obtained by the HRM analysis, when used to the embodiments able to distinguish different mutations of SARS-CoV-2, allows the rapid identification of mutations that can have a strong impact on disease severity or viral contagiousness. Indeed recently we have shown an association between certain prevalent mutations of SARS-CoV2, originated in Europe since mid-February 2020, and disease severity grade/ viral contagiousness. This has strong impacts on epidemiology consideration about pandemic containment as well as for specific vaccine development. Mutations in critical residues inside specific immunogenic epitopes can allow the virus to escape from the vaccine-generated immunity, as it occurs periodically for seasonal flu.

* * *

While the foregoing is directed to embodiments of the invention, other and further embodiments of the invention may be devised without departing from the basic scope thereof, and the scope thereof is determined by the claims that follow.

BIBLIOGRAPHY

1. Lai et al. Severe acute respiratory syndrome coronavirus 2: the epidemic and the challenges. Int J antimicrob Agents (2020). 2. Phan et al. Genetic diversity and evolution of SARS-CoV-2. Infect Genet

Evol (2020).

3. Spiteri et al. First cases of coronavirus disease 2019 in the WHO European Region. Euro Surveill (2020).

Claims

1. A molecular fingerprinting method to detect and genotype RNA targets in a sample through reverse transcriptase polymerase chain reaction (RT-PCR), said method being able to detect, discriminate and genotype different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations, said method comprising:

- performing, at the end of the RT-PCR amplification, a High Resolution Melting (HRM) analysis on the RT-PCR reaction mixture and the sample previously subjected to RT-PCR amplification; wherein the RT-PCR reaction mixture comprises one or more reverse transcription primers for reverting one or more RNA target molecules into cDNA molecules, wherein the RT-PCR reaction mixture comprises two or more pairs of amplification primers for amplifying in a multiplex approach two or more cDNA molecules, wherein said amplification primers are designed in order to generate amplicons with a different melting temperature each other in order to discriminate, in the HRM analysis, each amplicon by observing the specific melting temperature of each amplicon, wherein the method further comprises monitoring, during the HRM analysis, the change in the signal emission resulting from the temperature-induced denaturation of the double- stranded amplicons into two single-stranded DNAs, due to the release of the intercalating molecule or compound, wherein the method further comprises determining discrimination and genotyping of different strains or mutations of the same pathogen, different pathogens belonging to separated genus and genetic variations in the sample, through a reader analysing the signal variation and obtaining the result of the analysis through a graphic interface connected to said reader.

2. The method according to claim 1, wherein said amplification primers are designed to amplify pathogen RNA reverted cDNA targets producing corresponding amplicons, each from 38 to 1500 bps.

3. The method according to claim 1 or 2, wherein the amplification primers are designed to amplify an amplicon, wherein the melting peak of the amplicon is between 65 and 95°C.

4. The method according to claim 1, 2 or 3, wherein each amplification primer is present in the RT-PCR reaction mixture at a final concentration range from 50 to 1000 nanomolar (nM).

5. The method according to any of claims 1 to 4, wherein the reverse transcription and amplification buffer of the RT-PCR reaction mixture further comprises a DNA polymerase, a reverse transcriptase, deoxynucleoside triphosphates (dNTPs) or analogues, a buffer solution, water, a source of monovalent or bivalent cations, bovine serum albumin (BSA).

6. The method according to claim 5, wherein the amplification buffer of the RT- PCR reaction mixture further comprises additives selected between one, more or all of additives in a group comprising: NP40, DMSO, TMAC (Tetramethylammonium Chloride), Acetamide, Triton, Formamide, Betaine, E. Coli ssDNA binding protein, Glycerol, L-Camitine and Gelatine.

7. The method according to any of claims 1 to 6, wherein performing RT- PCR amplification using said RT-PCR reaction mixture includes amplifying the target purified nucleic acid using said RT-PCR reaction mixture to generate an amplicon, wherein amplifying the target purified nucleic acid includes thermocycling by repeatedly performing a ramp of temperature, wherein, during thermocycling in the RT-PCR amplification, monitoring the change in the signal emission resulting from the temperature-induced denaturation of the double- stranded amplicons or amplification products into two single-stranded DNAs, due to the release of the intercalating molecule or compound, is performed, wherein said ramp of temperature comprises performing the following temperature steps: reverse transcription at 40-65°C from 1 minute to 50 minutes denaturation at 95-98°C from 1 second to 30 seconds; annealing in a range between 50°C and 70°C from 1 second to 60 seconds; extension in a range between 60°C and 75°C from 0 second to 5 minutes.

8. The method according to any of claims 1 to 7, wherein performing HRM analysis includes performing a ramp of temperature for HRM on the RT-PCR reaction mixture previously subjected to RT-PCR amplification and, during the ramp of temperature, performing said monitoring the change in the signal emission resulting from the temperature-induced denaturation of the double- stranded amplicons into two single-stranded DNA, due to the release of the intercalating molecule or compound, wherein said ramp of temperature comprises includes performing the following temperature steps: incubation at 95 °C from 1 second to 60 seconds incubation at 60°C from 30 seconds to 2 minutes ramping from 65 °C up to 95 °C increasing the temperature 0.1°C/second or less.

9. The method according to any of claims 1 to 8, wherein the RT-PCR amplification occurs in a real-time PCR machine or in a thermocycling machine able to acquire said signal emission each 0.1°C/second or less.

10. The method according to any of claims 1 to 9, wherein said RT-PCR amplification is performed in a thermocycler or in a real-time PCR thermocycler, wherein, when the PCR amplification is performed in a thermocycler, HRM analysis is performed in a separate instrument, while when the RT-PCR amplification is performed in a real-time PCR thermocycler, HRM analysis is performed in the same real-time PCR thermocycler.

11. The method according to any of claims 1 to 10, wherein said RT-PCR reaction mixture is loaded periodically with a melting temperature calibrator to check the effective melting temperature of the amplicons of a particular thermocycler machine, and compare it with an expected melting temperature, wherein said calibrator is composed by synthetic oligonucleotides corresponding to the amplicons generated by the specific primers of the RT-PCR reaction mixture.

12. The method according to any of claims 1 to 11, wherein two or more sets of primers are further used that are specific for the same RNA targets, wherein a first set is present in a PCR reaction mixture in one well, at least a second set is present in another well, wherein each set of primers comprises primers each recognizing one specific RNA target, wherein the RNA targets recognized by the first set of primers are the same as the RNA targets recognized by the second set of primers, wherein the melting temperature of an amplicon generated by a primer of one set of primers recognizing a specific RNA target is different from the melting temperature of an amplicon generated by a primer of the other set of primers recognizing said specific RNA target, wherein, for each target RNA, HRM data are collected for each well, such that each target RNA is defined for several melting peaks, one for each well.

13. The method according to any of claims 1 to 12, wherein the pathogen is an infectious pathogen able to cause respiratory diseases.

14. The method according to claim 13, wherein the pathogen is Severe Acute Respiratory Syndrome - CoronaVirus - 2 (SARS-CoV-2).

15. The method according to claim 13, wherein said amplification primers include one or more specific primers for different loci of said pathogen that, preferably, contain “hotspot site” for mutation(s).

16. The method according to claim 15, wherein said specific primers for each of said loci generate amplicons that have differences in melting temperature not exceeding 30°C.

17. The method according to claim 14 wherein said amplification specific primers are able to amplify and identify, through HRM analysis, amplicons containing the following SARS CoV-2 genome (NCBI Reference Sequence: NC 045512.2) mutation positions: 155, 883, 1189, 1397, 3036, 8782, 9438, 11083, 14408, 21767, 23403, 25320, 25653, 26143, 27045, 28144, 28688, 28881, 29095, 2891, 4654, 9438, 27045, 28688, 29084.

18. The method according to claim 13, wherein the pathogen belongs to the coronaviridae family and/or influenza viruses family.

19. The method according to claim 18, wherein the pathogen is SARS-CoV, SARS-CoV-2, MERS-CoV, Influenzae virus A, B, C, D.

20. The method according to any of claims 1 to 19, wherein one further set of normalizing primers are further used, said amplification of human RNA serving as an internal RT-PCR validation control and/or control for normalization of the amplified pathogen RNA obtained.

21. The method according to claim 20, wherein said pathogen is SARS-CoV-2 and a pair of said normalizing primers targets a fragment from the human RNAse P transcript.

22. The method according to any of claims 1 to 21, wherein said sample is a crude sample, in particular said crude sample can be nasopharyngeal swabs, oropharyngeal swabs, buccal swabs, sputum, saliva, nasal swabs, vaginal or cervical mucus, other bodily fluids, blood, urine, biopsies, formalin-fixed paraffin-embedded ( FFPE ) tissue, cells, fine needle aspiration biopsies or similar.

23. A diagnostic kit to detect and genotype DNA targets, said kit comprising a Reverse-Transcription Polymerase Chain Reaction (RT-PCR) reaction mixture used for performing in at least one reaction a Reverse Transcription coupled with PCR amplification and a subsequent a High Resolution Melting (HRM) analysis on the RT-PCR reaction mixture previously subjected to RT-PCR, wherein said RT-PCR reaction mixture comprises one or more reverse transcription primers for reverting one or more RNA target molecules into cDNA molecules and two or more pairs of amplification primers for amplifying in a multiplex approach two or more cDNA molecules, wherein said amplification primers are designed in order to generate amplicons with a different melting temperature each other in order to discriminate, in the HRM analysis, each amplicon by observing the specific melting temperature of each amplicon.

24. The diagnostic kit according to claim 23, wherein said amplification primers are designed to amplify pathogen RNA reverted cDNA targets producing corresponding amplicons, each from 38 to 1500 bps.

25. The diagnostic kit according to claim 23 or 24, wherein the amplification primers are designed to amplify an amplicon, wherein the melting peak of the amplicon is between 65°C and 95°C.

26. The diagnostic kit according to claim 23, 24 or 25, wherein each amplification primer is present in the RT-PCR reaction mixture at a final concentration range from 50 to 1000 nanomolar (nM).

27. The diagnostic kit according to any of claims 24 to 26, wherein said RT- PCR reaction mixture comprises an amplification buffer comprising a reverse transcriptase, DNA polymerase, deoxynucleoside triphosphates (dNTPs) or analogues, a buffer solution, water, a source of monovalent or bivalent cations, bovine serum albumin (BSA).

28. The diagnostic kit according to claim 27, wherein said amplification buffer further comprises additives selected between one, more or all of additives in a group comprising: NP40, DMSO, TMAC (Tetramethylammonium Cloride), Acetamide, Triton, Formamide, Betaine, E. Coli ssDNA binding protein, Glycerol, L-Camitine and Gelatine.

29. The diagnostic kit according to any of claims 23 to 28, wherein the pathogen is a pathogen responsible for respiratory diseases.

30. The diagnostic kit according to claim 29, wherein the pathogen is Severe Acute Respiratory Syndrome - CoronaVirus - 2 (SARS-CoV-2).

31. The diagnostic kit according to any of claims 23 to 30, wherein said amplification primers include one or more specific primers for different RNA reverted cDNA targets corresponding to loci of said pathogen, that, preferably, contain “hotspot site” for mutation(s) .

32. The diagnostic kit according to claim 31, wherein said amplification primers include one or more specific primers able to distinguish different mutant strains of SARS-CoV-2, associated with different disease severity grades or viral contagiousness.

33. The diagnostic kit according to claim 31 or 32, wherein said specific primers for each genotype generate amplicons that have differences in melting temperature not exceeding 30°C.

34. The diagnostic kit according to claim 29, wherein the pathogen belongs to the coronaviridae family and/or influenza virus family.

35. The method according to claim 34, wherein the pathogen is SARS-CoV, SARS-CoV-2, MERS-CoV, Influenza virus A, B, C, D.

36. Amplification primers for performing a molecular fingerprinting method to detect and genotype pathogens RNA reverted cDNA targets, wherein the method is able to discriminate and genotype different strains of the same pathogen, different pathogens belonging to separated genus and genetic variations/mutations in a single reaction, wherein said amplification primers are provided for amplifying in a multiplex approach two or more target nucleic acids in a RT-PCR amplification, wherein said primers are designed in order to generate amplicons with a different melting temperature each other in order to discriminate, in a High Resolution Melting (HRM) analysis following the RT- PCR amplification, each amplicon by observing the specific melting temperature of each amplicon.

37. The amplification primers according to claim 36, wherein the pathogen is a pathogen responsible for a respiratory disease.

38. The amplification primers according to claim 37, wherein the pathogen is Severe Acute Respiratory Syndrome - CoronaVirus - 2 (SARS-CoV-2).

39. The amplification primers according to any of claims 36 to 38, wherein said amplification primers include one or more specific primers for different RNA reverted cDNA targets corresponding to loci of said pathogen, that, preferably, contain “hotspot site” for mutation(s).

40. The amplification primers according to claim 39, wherein said specific primers for each genotype generate amplicons that have differences in melting temperature not exceeding 30°C.

41. The amplification primers according to claim 37, wherein the pathogen belongs to the coronaviridae family and/or influenza virus family.

42. The amplification primers according to claim 41, wherein the pathogen is SARS-CoV, SARS-CoV-2, MERS-CoV, Influenza virus A, B, C, D.

43. A method to design therapeutic small molecules capable to defeat SARS CoV-2 infection or decrease viral infectivity and pathogenicity through the interaction of those molecules with specific protein epitopes whose shape is influenced and modulate by the presence one or more of the said RNA SARS CoV-2 genome mutations by detection and genotyping of viral mutations using a method according to any of claims 1-22.

44. A method to design specific biological drugs capable to defeat SARS CoV- 2 infection or decrease viral infectivity and pathogenicity through the interaction of those molecules with specific protein epitopes whose shape is influenced and modulate by the presence one or more of the said RNA SARS CoV-2 genome mutations by detection and genotyping of viral mutations using a method according to any of claims 1-22.

45. A method to design specific therapeutic small RNA, short Hairpin RNA, microRNA, siRNAs capable to target RNA viral genome of SARS CoV-2 triggering its genome degradation by detection and genotyping of viral mutations using a method according to any of claims 1-22.

46. A method to design vaccines, capable to prevent or defeat SARS CoV-2 infection through the interaction of antibodies, raised consequently patient vaccination, with specific protein epitopes whose shape is influenced and modulate by the presence one or more of the said RNA SARS CoV-2 genome mutations by detection and genotyping of viral mutations using a method according to any of claims 1-22.

47. An apparatus for performing a molecular fingerprinting method to detect and genotype pathogens RNA-reverted cDNA targets through Reverse Transcriptase coupled in one step with Polymerase Chain Reaction (PCR) is provided, wherein said method is able to discriminate and genotype different strains/mutations/loci of the same pathogen, different pathogens belonging to separated genus and genetic variations in a single reaction, wherein said apparatus comprises:

- a RT-PCR amplification device configured for using said RT-PCR reaction mixture and said sample;

- a device for performing a HRM analysis on the RT-PCR reaction mixture and the sample previously subjected to RT-PCR amplification; wherein the RT-PCR reaction mixture comprises one or more reverse transcription primers for reverting, one or more RNA target molecules into cDNA molecules, wherein the RT-PCR reaction mixture comprises two or more pairs of amplification primers for amplifying in a multiplex approach two or more cDNA molecules, wherein said amplification primers are designed in order to generate amplicons with a different melting temperature each other in order to discriminate, in the HRM analysis, each amplicon by observing the specific melting temperature of each amplicon;

- monitoring means for monitoring, during the HRM analysis, the change in the signal emission resulting from the temperature-induced denaturation of the double- stranded amplicons into two single-stranded DNA, due to the release of the intercalating molecule or compound,