EP4118232A1 - Procédés d'empreinte moléculaire pour détecter et génotyper différentes cibles d'arn par réaction en chaîne de la polymérase à transcription inverse en une seule réaction - Google Patents

Procédés d'empreinte moléculaire pour détecter et génotyper différentes cibles d'arn par réaction en chaîne de la polymérase à transcription inverse en une seule réaction

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Publication number
EP4118232A1
EP4118232A1 EP20716077.1A EP20716077A EP4118232A1 EP 4118232 A1 EP4118232 A1 EP 4118232A1 EP 20716077 A EP20716077 A EP 20716077A EP 4118232 A1 EP4118232 A1 EP 4118232A1
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European Patent Office
Prior art keywords
amplification
primers
pcr
pathogen
rna
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EP20716077.1A
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German (de)
English (en)
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Rudy IPPODRINO
Maria PACHETTI
Elisabetta MAURO
Bruna MARINI
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Ulisse Biomed SpA
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Ulisse Biomed SpA
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Publication of EP4118232A1 publication Critical patent/EP4118232A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6846—Common amplification features
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • 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.
  • RT-PCR Reverse Transcription Polymerase Chain Reaction
  • 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.
  • SARS-CoV Severe Acute Respiratory Syndrome - CoronaVirus
  • SARS-CoV-2 SARS-CoV-2 causing the COVID-19 disease
  • MERS-CoV Middle East Respiratory Syndrome - CoronaVirus
  • Influenza viruses through RT-PCR using High Resolution Melting (HRM) technology in multiplex assay format.
  • HRM High Resolution Melting
  • RT-PCR Reverse transcription Polymerase Chain Reaction PCR
  • RNA rather than DNA as starting template.
  • 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.
  • DNA polymerase is used to convert the single-stranded cDNA into double-stranded DNA.
  • High Resolution Melting 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.
  • SNPs single nucleotide polymorphisms
  • 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.
  • 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.
  • 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.
  • the diagnostic assays include a reverse transcription step followed by a PCR with primers able to amplify specific parts of the viral genome.
  • RT-PCR reverse-transcriptase polymerase chain reaction coupled
  • 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.
  • 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.
  • the method includes:
  • 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
  • the RT-PCR reaction mixture comprises one or more reverse transcription primers for reverting one or more RNA target molecules in cDNA molecules
  • 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
  • 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
  • a diagnostic kit for detection and genotyping of RNA targets 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the apparatus includes:
  • 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;
  • PCR amplification device configured for using said PCR reaction mixture and said sample
  • the RT-PCR reaction mixture comprises one or more reverse transcription primers for reverting, in a multiplex approach, two or more target nucleic acids
  • 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.
  • the RNA target that can be detected and genotyped is a pathogen RNA target.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • inventions 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).
  • 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:
  • 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;
  • 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);
  • embodiments of the present disclosure can be executed by any real-time PCR machine and do not require a dedicated and specific instrument;
  • 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.
  • 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.
  • 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.
  • FIG. 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.
  • 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.
  • the method includes:
  • 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
  • the RT-PCR reaction mixture comprises one or more reverse transcription primers for reverting one or more RNA target molecules into cDNA molecules
  • 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
  • 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 strain
  • the RNA target that can be detected and genotyped is a pathogen RNA target.
  • the pathogen is an infectious pathogen able to cause a respiratory diseases
  • the pathogen is SARS-CoV2.
  • the amplification primers include one or more specific primers for different loci of the pathogen that, preferably, contain “hotspot site” for mutation(s).
  • an embodiment is able to detect and genotype SARS-CoV2 strains RNA.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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
  • 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
  • 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;
  • 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.
  • 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.
  • 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.
  • the amplification primers are sufficiently complementary to the target nucleic acid to hybridize therewith and trigger polymerase-mediated synthesis.
  • 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).
  • the amplification primers are designed to amplify an amplicon wherein the melting peak of the amplicon is between 65°C and 95°C.
  • 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.
  • the amplification primer is fully matched to cDNA template the amplification proceeds with full efficiency.
  • 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.
  • each amplification primer is present in the RT-PCR reaction mixture at a final concentration range from 50 to 1000 nanomolar (nM).
  • the RT-PCR reaction mixture comprises the reverse transcription primers, the amplification primers and the reverse transcription and amplification buffer.
  • the reverse transcription and amplification buffer is comprised in a diagnostic kit that is part of the present disclosure.
  • 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.
  • the polymerase that can be used in association with embodiments described herein is a hot-start polymerase.
  • 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.
  • the reverse transcriptase used can be Superscript IV One-step RT-PCR (Thermo Fischer Scientific), TaqPathTM 1-Step Multiplex Master Mix, Power SYBR® Green RNA-to-CTTM 1-Step, EXPRESS One-Step SYBRTM GreenERTM, Maxima H Minus cDNA Synthesis Master Mix.
  • 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.
  • 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.
  • 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.
  • the intercalating molecule or compound can be an intercalating dye emitting fluorescence.
  • 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.
  • 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.
  • the buffer solution may comprise water, in particular deionized water, TrisHCl and/or KC1 and possibly in some cases MgCl 2 .
  • the RT-PCR reaction mixture may further comprise a pH stabilizer.
  • the RT-PCR reaction mixture may further comprise preservatives.
  • the RT-PCR reaction mixture may further comprise water.
  • 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.
  • a chloride containing monovalent ion or bivalent ions can be used.
  • potassium ions can be used as a source of monovalent cations.
  • K + can be obtained from potassium salts, e.g. potassium chloride, in particular potassium chloride at a concentration of about 0.1 M.
  • magnesium or manganese ions can be used as a source of bivalent cations.
  • Mg can be obtained from magnesium salts, e.g. magnesium chloride.
  • the RT-PCR reaction mixture may further comprise bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the RT-PCR reaction mixture further comprises one or more detergents.
  • said detergent can be Nonidet-P40 (NP40) at a concentration from 0.1 to 1%.
  • the RT-PCR reaction mixture may further comprise additives.
  • the additives can be included in some embodiments of the above-mentioned amplification buffer.
  • 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.
  • the presence of additives can be important to avoid a high basal fluorescence background allowing an increased diagnostic sensitivity, specificity and accuracy.
  • the additives may further comprise a gelatin, for example at a concentration of about 0.1%.
  • the additives may further comprise an enhancer.
  • the enhancer can be L-Camitin at a concentration of about 0,42 M.
  • the additives may further comprise sugar alcohol, for example sorbitol at a concentration of about 25mM.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • amplicons are generated in which the intercalating molecule or compound binds in the extension step, during which the signal (e.g. fluorescence) is acquired.
  • 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.
  • 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.
  • the RT-PCR amplification may occur in a thermocycling machine able to acquire said signal emission each 0.1°C/second or less.
  • the HRM analysis includes performing a ramp of temperature on the RT- PCR reaction mixture previously subjected to RT-PCR amplification.
  • a ramp of temperature includes performing the following temperature step:
  • 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.
  • low temperature e.g. 60°C
  • amplicons are all double-stranded and the maximum level of signal, e.g. fluorescence, is detected.
  • 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.
  • the amplification primers designed according to the present disclosure allow to amplify amplicons with a precise and specific melting temperature fingerprint each.
  • 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.
  • a pathogen or a group of pathogens e.g. a virus
  • the real-time PCR 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.
  • RT-PCR amplification can be for instance performed in a PCR thermocycler.
  • RT-PCR amplification can be typically performed in a real-time PCR machine, for instance a real-time PCR thermocycler.
  • the whole method can be performed in a single apparatus, in particular a real-time PCR machine.
  • 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.
  • the two operations i.e. RT-PCR amplification and HRM analysis
  • the two operations 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • a possible alternative second amplification buffer for diagnostic purposes 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.
  • the second amplification buffer can be used as a PCR enhancer buffer providing increased diagnostic sensitivity, specificity and accuracy as above discussed.
  • 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).
  • MAC Tetramethylammonium Chloride
  • Acetamide final concentration range: from 0% to 10%
  • Formamide final concentration range: from 0% to 10%
  • Betaine final concentration range: from 0 mM to 8 mM
  • Gelatine final concentration range: from 0 mg/ml to 3.5 mg/ml
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • each amplification primer is present in the RT-PCR reaction mixture at a final concentration range from 50 to 1000 nM.
  • 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.
  • specific primers for the most conserved loci of the viral genome are provided in the RT-PCR reaction mixture of the present disclosure.
  • 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.
  • 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)
  • 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) MgCl 2 (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).
  • dNTPs final concentration 0.2mM
  • MgCl 2 final concentration 0.75mM
  • TrisHCl buffer solution final concentration 30mM: and pH 9.0
  • KC1 final concentration 50mM
  • BSA final concentration lOpg/ml
  • DNA polymerase g) Reverse Transcriptase h )SYTO-9 (final concentration 4mM).
  • a further alternative possible specific amplification buffer for SARS-CoV-2 diagnosis 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).
  • TMAC Tetramethylammonium Cloride
  • Acetamide final concentration range: from 0% to 10%
  • Formamide final concentration range: from 0% to 10%
  • Betaine final concentration range: from 0 mM to 8 mM
  • Gelatine final concentration range: from 0 mg/ml to 3.5 mg/ml
  • dNTPs final concentration 0.15 mM
  • MgCl 2 final concentration 0.75 mM
  • TrisHCl buffer solution final concentration 30mM; pH 9
  • KC1 final concentration: 40mM
  • BSA final concentration 10pg/ml
  • DNA polymerase g) reverse transcriptase
  • SYTO-9 final concentration 2 mM
  • SYTO-9 final concentration 2 mM
  • SYTO-9 final concentration 2 mM
  • SYTO-9 final concentration 2 mM
  • SYTO-9 final concentration 2 mM
  • TMAC Tetramethylammonium Chloride, final concentration of 75 mM
  • Acetamide final concentration 3%)
  • Formamide final concentration 1,5%)
  • Betaine final concentration 0,5M
  • Gelatine final concentration 0,lmg/mL
  • the diagnostic SARS-CoV-2 test based on the embodiments described herein can provide important information about SARS-CoV-2 spread and prognosis:
  • 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.
  • 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.
  • 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.

Abstract

Des modes de réalisation de la présente divulgation concernent des amorces, des oligonucléotides, des composés chimiques et des procédés pour une analyse par réaction en chaîne par polymérase à transcription inverse (RT-PCR) multiplex capable d'effectuer la détection et le génotypage de différentes souches d'ARN du même agent pathogène, ou de différents agents pathogènes appartenant à des genres ou familles séparés, et de variations génétiques en une seule réaction.
EP20716077.1A 2020-03-13 2020-03-13 Procédés d'empreinte moléculaire pour détecter et génotyper différentes cibles d'arn par réaction en chaîne de la polymérase à transcription inverse en une seule réaction Pending EP4118232A1 (fr)

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US20210155973A1 (en) * 2017-06-20 2021-05-27 Ulisse Biomed S.P.A. Methods for dna targets detection directly in crude samples through polymerase chain reaction and genotyping via high resolution melting analysis
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