New method of detection of several target nucleic acids in a biological sample

Abstract

The present invention concerns an in vitro method of detecting at least two target nucleic acid sequences from a biological sample comprising: a) releasing and / or extracting the nucleic acids from the biological sample, and b) amplifying and detecting at least two target nucleic acid sequences in a PCR reaction using the nucleic acids of step a) as a template, wherein the DNA amplification and detection step b) comprises: (b1) hybridizing each target nucleic acid sequence with a specific primers pair composed of a forward primer and a reverse primer comprising nucleotide sequences complementary to the target nucleic acid sequence, wherein at least one primer of the pair additionally comprises a tag sequence that is linked to its 5'-end portion; (b2) contacting the resultant of step b1) with a template-dependent nucleic acid polymerase under primer extension conditions for inducing the 3'-extension reaction and forming an extended neosynthetized strand; (b3) denaturing the amplified product of step (b2) and contacting the extended neosynthetized single-strand with a probe sequence comprising a 3' blocker and an interactive label, wherein the probe sequence hybridizes with the 3'-end portion of the extended neosynthetized single-strand and forms, under conditions for inducing the 3'-extension reaction, an extended duplex providing the signal; and c) optionally quantifying the amount of the said target nucleic acids.

Description

[0001] New method of detection of several target nucleic acids in a biological sample

[0002] TECHNICAL FIELD OF THE INVENTION

[0003] The present invention relates to the field of technologies for the detection of nucleic acids in a biological sample, and in particular multiplex detection of nucleic acids from a single biological sample.

[0004] BACKGROUND ART

[0005] Nucleic acid detection techniques, with or without amplification, have been developed in many fields of biology. In the majority of cases, these techniques consist of monitoring the amplification of the signal in real time. Fluorescent probes are generally used to hybridize with the target nucleic acids. These detection methods therefore include all technologies using labelled probes such as TaqMan hydrolysis probes (Pereira et al., 2014; Liu et al., 2019) or probes called "Molecular beacon" (Gatcombe et al. 2010; Salvatore et al. 2021). With the best detection tools on the market, these technologies can discriminate up to six targets using six distinct fluorescence channels. Therefore, these techniques are limited to the detection of one pathogen per fluorescence channel on the same biological sample. Other technologies allow the detection of several amplification curves on the same fluorescence channel, but these technologies are very complicated to develop and analyse, and increase the time required to process the results.

[0006] As a result, new multiple detection techniques have emerged. The most promising ones use an intercalating dye that emits fluorescence when it is integrated inside a double-stranded PCR product (Reed 2007; Phung et al, 2013). This type of technology is most often associated with a DNA fusion step, allowing the visualisation of fluorescence profiles specific to each PCR product. Then, it is possible to detect up to twenty targets on a single fluorescence channel, which gives it a clear advantage over conventional real-time PCR technologies. However, only one fluorescence channel can be used for nucleic acid fusion, which somewhat limits the amounts of targets that can be detected. In addition, these DNA fusion methods are sensitive to non-specific signals and are dependent on genetic variations within the target sequences. It is therefore essential to limit the number of detected pathogens to not affect the interpretation of the results. So, there is still a need to provide a new method of detection of several target nucleic acid sequence(s) from a biological sample, that would be not sensitive to non-specific signals nor be dependent on genetic variations within the target sequences and that would permit the simultaneous detection of several target sequences in a single biological sample.

[0007] The Applicant specifically demonstrated that the use of a tag sequence that is linked to the 5’-end portion of a primer (forward or reverse of a primers pair, or both) in a PCR amplifying reaction, and a specific probe sequence having a 3’ blocker and hybridizing the 3’-end portion of the extended neosynthetized single-strand (which is the complementary sequence of the said tag sequence), permits to detect at least two target nucleic acid sequences from a biological sample with several advantages (schema Figure 10). Indeed, the method of the invention makes the detection of a target completely independent of its composition or nucleotide length. Thus, the fluorescence signal is not modified by variations in the target sequence. It is then possible to detect several target sequences (ie ‘multiplex’) on the same fluorescence channel, without interpretation errors due to variations or mutations in the target. The method of the invention also provides the ability to multiply the detection on several channels (e.g 6 to 8 signals per channel), which increases the target multiplexing capacity. The method of the invention can also be combined with combination with a realtime PCR detection using a TaqMan probe. Therefore, different targets can be detected in the same well using these two different technologies. This allows to offer increased multiplexing, as well as the possibility to specifically quantify a target if required.

[0008] Advantageously, in comparison to usual hybridization between the probe and the target sequence disclosed in the prior art, the method of the invention provides more flexibility on the design of the probe, in terms of length, concentration in GC, Tm and is particularly adapted to multiplex embodiments.

[0009] So, this makes it possible to reduce the cost and analysis time per sample tested.

[0010] The method of the invention can be used in many domains of molecular biology, such as for detecting the presence or level of target sequence(s) (eg genes or molecular markers) in a biological sample (eg animal, human, plant, environmental sample...), and / or screening and selecting genes or markers of interest (eg genetic traits, phenotypic traits), notably in the domains of health, virology, agriculture, ecological or environmental field.

[0011] SUMMARY OF THE INVENTION

[0012] So, a first object of the invention is an in vitro method of detecting at least two target nucleic acid sequences from a biological sample comprising: a) releasing and / or extracting the nucleic acids from the biological sample, and b) amplifying and detecting at least two target nucleic acid sequences in a PCR reaction using the nucleic acids of step a) as a template, wherein the DNA amplification and detection step b) comprises:

[0013] (b1) hybridizing each target nucleic acid sequence with a specific primers pair composed of a forward primer and a reverse primer comprising nucleotide sequence complementary to the target nucleic acid sequence, wherein at least one primer additionally comprises a tag sequence that is linked to its 5’-end portion;

[0014] (b2) contacting the resultant of step b1) with a template-dependent nucleic acid polymerase under primer extension conditions for inducing the 3’-extension reaction and forming an extended neosynthetized strand;

[0015] (b3) denaturing the amplified product of step (b2) and contacting the extended neosynthetized single-strand with a probe sequence comprising a 3’ blocker and an interactive label, wherein the probe sequence hybridizes with the 3’-end portion of the extended neosynthetized single-strand and forms, under conditions for inducing the 3’- extension reaction, an extended duplex providing the signal; and

[0016] (c) optionally quantifying the amount of the said target nucleic acids.

[0017] The invention concerns an in vitro method of detecting at least two target nucleic acid sequences, for a multiplex analysis. The technology of the invention is named “MPM technology” for Multiplex Probe Melting technology.

[0018] The invention also relates to a use of a kit in the in vitro method of the invention for detecting at least two target nucleic acid sequences from a biological sample, which comprises: a) a specific primers pair for each target nucleic acid sequence, wherein at least one primer of the pair additionally comprises a tag sequence that is linked to its 5’-end portion; b) a template-dependent nucleic acid polymerase capable of inducing the 3’-extension reaction at the 3’-end of the primer and forming an extended neosynthetized strand; c) at least one probe sequence comprising a 3’ blocker and an interactive label, wherein the probe sequence hybridizes with the 3’-en portion of the extended neosynthetized singlestrand ; d) optional reagents for denaturing the amplified products and performing target amplification PCR such as buffers, DNA polymerase cofactors, deoxyribonucleotide-5-triphosphates, and reverse transcriptase ; e) optional reagents necessary for performing positive and negative control reactions ; and f) optional reagents for detecting the extended duplex formed with the probe sequence comprising the interactive label.

[0019] The in vitro method according to the invention or the kit according to the invention will be generally used for detecting, and optionally quantifying, nucleotide variations or mutations, in particular variations or mutations correlated with genetic or phenotypic traits in a subject (for genotyping or screening applications) and / or correlated with physiological disorders of a subject (for detecting or monitoring a disorder).

[0020] As mentioned above, the in vitro method and the kit of the invention can be used in many domains of molecular biology, such as detecting the presence or level of target sequence(s) (eg genes or molecular markers) in a biological sample (eg animal, human, plant, environmental sample), and / or screening and selecting genes or markers of interest, notably in the domains of health, virology, agriculture, ecological or environmental field.

[0021] As examples, the method and kit of the invention can be used in the infectious diagnosis of several pathogens within the same sample, or to analyse mutations of interest within the same microbial strain, in particular for monitoring the epidemiology of microbial outbreaks.

[0022] The method and kit of the invention can also be used to detect any type of molecular markers, for example oncology markers, inflammation markers, and many others in a biological sample of a subject of interest.

[0023] It can also be used in genotyping or screening project, to select the microorganism’s gene, or to select seeds in the agricultural field. In this field using the method of the invention could also allow the specific detection of phytopathogen organism.

[0024] In a particular embodiment, the in vitro method according to the invention or the kit according to the invention are used for detecting and / or quantifying nucleic acids of pathogens having infected the subject or pathogens present in a sample of environment.

[0025] So, another object of the invention is use of the in vitro method according to the invention or use of the kit according to the invention for for (i) detecting and optionally quantifying nucleic acids target sequence(s) in a biological sample such as animal, human, plant, or environmental sample, and / or (ii) screening and selecting genes or markers of interest, notably in the domains of health, virology, agriculture, ecological or environmental field, in particular detecting and optionally quantifying, nucleotide variations or mutations in a subject, such as variations correlated with genetic or phenotypic traits of the subject , or variations or mutations correlated with physiological disorders of a subject, and / or detecting and optionally quantifying nucleic acids of pathogens having infected the subject or pathogens present in a sample of environment.

[0026] DESCRIPTION OF THE FIGURES

[0027] Figure 1: Comparison of melting profiles from a real-time PCR coupled with a melting step performed on synthetic DNA with the method of the invention, containing the tag on the forward primer, on the reverse primer or both.

[0028] Figure 2: Comparison of melting profiles from a real-time PCR coupled with a melting step, between a reaction mix with fluorescent intercalant and a reaction mix using the method of the invention, performed on different Influenza B DNA or RNA samples.

[0029] Figure 3: Comparison of the melting profiles resulting from a real-time PCR coupled to a melting step, between different reaction mixes using the method of the invention containing Melting Probe (MP) with a reporter and a quencher distant from 15 to 55 nucleotides, performed on synthetic DNA of Metapneumovirus.

[0030] Figure 4: Comparison of melting profiles from real-time PCR coupled to a melting step, between different reaction mixes using the method of the invention with different blockers on the 3' end of the MP, performed on synthetic Rhinovirus DNA.

[0031] Figure 5: Comparison of the fusion profiles from a real-time PCR coupled to a melting step, between an MP blocked in 3' with a C3 Spacer and quenched in 5' with a BHQ1 , and an MP blocked and quenched at the same time in 3' with a BHQ1, both performed with synthetic Rhinovirus DNA.

[0032] Figure 6: Presentation of the melting profiles obtained from a real-time PCR coupled with a melting step, performed from a duplex reaction mix for the detection of synthetic DNA of Rhinovirus and Metapneumovirus independently and simultaneously.

[0033] Figure 7: Melting profile obtained from a real-time PCR coupled with a melting step on the HEX channel, compared with the melting profile from the FAM channel, performed on Metapneumovirus synthetic DNA with the method of the invention.

[0034] Figure 8: Detection of synthetic Rhinovirus DNA using the technology of the invention on the FAM channel, coupled with detection of synthetic Metapneumovirus DNA using TaqMan realtime PCR on the CFR610 channel. Figure 9: Detection of synthetic DNA from Coronavirus OC43 with MPM technology using a tag that hybridizes completely (tag-neuf) or not (tag-sept) on the fluorescence probe in HEX channel.

[0035] Figure 10: Schema of the technology of the invention.

[0036] DETAILED DESCRIPTION OF THE INVENTION

[0037] So, the present invention relates to an in vitro method of detecting at least two target nucleic acid sequences from a biological sample comprising: a) releasing and / or extracting the nucleic acids from the biological sample, and b) amplifying and detecting at least two target nucleic acid sequences in a PCR reaction using the nucleic acids of step a) as a template, wherein the DNA amplification and detection step b) comprises:

[0038] (b1) hybridizing each target nucleic acid sequence with a specific primers pair composed of a forward primer and a reverse primer comprising nucleotide sequences complementary to the target nucleic acid sequence, wherein at least one primer of the pair additionally comprises a tag sequence that is linked to its 5’-end portion;

[0039] (b2) contacting the resultant of step b1) with a template-dependent nucleic acid polymerase under primer extension conditions for inducing the 3’-extension reaction and forming an extended neosynthetized strand;

[0040] (b3) denaturing the amplified product of step (b2) and contacting the extended neosynthetized single-strand with a probe sequence comprising a 3’ blocker and an interactive label, wherein the probe sequence hybridizes with the 3’-end portion of the extended neosynthetized single-strand and forms, under conditions for inducing the 3’- extension reaction, an extended duplex providing the signal; and

[0041] (c) optionally quantifying the amount of the said target nucleic acids.

[0042] By ‘quantifying’ the amount of the said target nucleic acids according to the invention, it encompasses measuring the amount of the said target nucleic acid (providing an indication of the quantity of the said target nucleic acid) or quantifying the amount of the said target nucleic acid by using a dilution range of DNA nearby. By ‘hybridizing each target nucleic acid sequence with a specific primers pair’ in step (b1), it means that we have a specific primers pair for each target nucleic acid sequence, in other words, we have as many specific primers pairs as target nucleic acid sequences. Indeed, a specific primers pair is capable of specifically binding to a target sequence and should not interfere with each other in order to amplify the target sequence.

[0043] METHOD OF DETECTING TARGET NUCLEIC ACIDS

[0044] Target nucleic sequences and biological sample

[0045] By ‘biological sample’ according to the invention, it encompasses a biological sample of a ‘subject of interest’ or a biological sample of environment.

[0046] In particular, the biological sample is selected in the group consisting of a sample from an animal or human subject, in particular a sample of bodily fluid such as blood and its derivatives, urine, saliva, faeces, milk, mucous secretions, nasopharyngeal or oropharyngeal swabs, bronchoalveolar fluids, trans-tracheal aspirates, a sample of tissue or an organ, or an eluate from rag; a sample derived from microbial culture, seeds or other plant tissues, or a sample of environment.

[0047] In particular, the ‘subject of interest’ is an animal, a human or a plant, infected or susceptible to be infected by a pathogen or a subject affected by a physiological or pathological disorder.

[0048] By ‘physiological or pathological disorder’, it means for example a subject with or at risk of developing a clinical condition such as cancer, an inflammatory disease, an infectious disease, or an autoimmune disease.

[0049] As used herein ‘nucleic acids’ or ‘nucleic acids sequences’ according to the invention refers to nucleotide polymers composed of deoxyribonucleotides, ribonucleotides, or a mixture of both. In a particular embodiment, the nucleic acid used in the method of the invention is desoxyribonucleic acid (DNA), preferably genomic DNA. Alternatively, the nucleic acid is ribonucleic acid (RNA).

[0050] By ‘target nucleic acids sequences’, it encompasses nucleic acids sequences of interest from the genome of different animal species (including human), microorganisms or plant species, notably selecting from the group consisting of :

[0051] (i) nucleic acids sequences correlated to genetic or phenotypic treats of interest (for genotyping or screening applications as examples), (ii) mutations or variations of nucleic acids sequences correlated to a physiological or pathological disorder (for detecting a disorder as example),

[0052] (iii) nucleic acids sequences of a pathogen that may have infected a subject or a sample of environment (for detecting an infection by a pathogen as example),

[0053] (iv) and mixtures thereof.

[0054] In particular, nucleic acids sequences correlated to genetic or phenotypic treats of interest encompass nucleic acids from non-pathogenic micro-organism or seeds which have genetic interest for different application such as health, agricultural, ecological or environmental field.

[0055] So, in a particular embodiment, the target nucleic acids in the method of the invention are nucleic acids sequences of interest from the genome of different animal species (including human), microorganisms or plant species selected from the group consisting of (i) nucleic acids sequences correlated to genetic or phenotypic treats of interest, mutations or variations of nucleic acids sequences correlated to a physiological or pathological disorder, (iii) nucleic acids sequences of a pathogen that may have infected a subject or a sample of environment, and mixtures thereof. In a particular embodiment, the target nucleic acids in the method of the invention are genomic nucleic acids of the subject (eg animal, human, microorganism or plant) and / or nucleic acids of pathogens having infected the subject or nucleic acids of pathogens present in a sample of environment or a sample of plant, in particular nucleic acids of virus, bacteria, fungus, yeast, parasite or mixtures thereof. A ‘pathogen’ as used herein is an organism associated with, or causative of, a disease or condition of animals or humans. Non-limiting examples of pathogens may include viruses inclusive of RNA and DNA viruses, bacteria, fungi, yeast and parasite. The technology of the invention is applicable to all pathogens, whatever the type, specie and genus of the pathogen.

[0056] In case of nucleic acids of a pathogen, for example, the genetic material of bacteria is usually DNA. Likewise, some viruses contain DNA, which may be either single-stranded or doublestranded (e.g., adenovirus, cytomegalovirus, polyomavirus, etc.). Other viruses contain RNA, either single-stranded (e.g., coronaviruses, hepatitis A viruses, influenza viruses, etc.) or double-stranded (e.g., rotaviruses).

[0057] Viruses whose nucleic acids can be detected by the method described herein are notably selected in the group consisting of: adenoviruses, astroviruses, hepatitis A and E viruses, rotavirus, norovirus and other caliciviruses, enteroviruses, polyomaviruses, cytomegalovirus, influenza viruses and other respiratory viruses. The respiratory viruses notably cover respiratory syncytial virus, parainfluenza viruses, metapneumovirus, and coronavirus, including the agents of SARS, respiratory adenoviruses, rhinovirus, and bocavirus. Bacteria whose nucleic acids can be detected by the present method are notably selected in the group consisting of: Salmonella spp., Yersinia spp., Listeria monocytogenes, Taylorella equigenitalis, Mycoplasma gallisepticum / Mycoplasma synoviae, Trichinella spp., Mastitis causing bacteria, such as e.g., Escherichia coli, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus uberis, Staphylococcus aureus, Staphylococcus chromogenes, Staphylococcus epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniaie, Cory nebacteri urn bovis, Mycobacterium bovis, Serratia marcescens, Trueperella pyogenes, Salmonella spp, Listeria Monocytogenes, Leptospira spp, Campylobacter fetus spp, Coxiella burnetii, Anaplasma phagocytophylum, Chlamydia spp, Brucella spp, Legionella pneumophila, and other Gram-positive and catalase-negative cocci, and mixtures thereof.

[0058] Fungi whose nucleic acids can be detected by the present method include Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Aspergillus niger, Trichophyton spp, Fusarium, Scedosporium, Alternaria, Exophiala, Histoplasma, Coccidioides, Penicillium marneffei, Histoplasma capsulatum, Blastomyces dermatitidis, Coccidioides spp., Paracoccidioides brasiliensis, Cryptococcus neoformans, Aspergillus fumigatus, Rhizopus, Lichtheimia, Rhizomucor, and mixtures thereof.

[0059] Yeast whose nucleic acids can be detected by the present method include Candida albicans.

[0060] Parasite whose nucleic acids can be detected by the present method include Toxoplasma gondii, Neospora caninum, Dirofilaria immitis, and mixtures thereof.

[0061] In a particular embodiment of the invention, the pathogen is a virus, in particular selected from respiratory virus such as influenza virus, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, and bocaviruses, and mixtures thereof, or a Human Immunodeficience Virus (HIV).

[0062] In another particular embodiment, the pathogen is a bacteria strain such as bacterial strains causing bovine mastitis, in particular Staphylococcus aureus, Streptococcus uberis, Escherichia coli, coagulase-negative Staphyloccus such as S. chromogenes, S. epidermidis, S. haemolyticus, S. hominis, Streptococcus agalactiae, Streptococcus dysgalactiae, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniaie, Cory nebacteri urn bovis, Mycobacterium bovis, Serratia marcescens, Trueperella pyogenes.

[0063] So, in a particular embodiment, the target nucleic acids in the method of the invention are DNA or RNA of a virus, in particular selected from respiratory virus such as influenza virus, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, and bocaviruses, or a Human Immunodeficience Virus (HIV), or DNA of bacteria strain such as bacterial strains causing bovine mastitis.

[0064] The target nucleic acids may be released from the biological sample or may be extracted from the biological sample.

[0065] By ‘released from’, it means that the nucleic acids are simply released from the biological sample, without extraction and purification steps. Nucleic acids can be released from a sample by heating at high temperature (eg: 95-98°C) and / or by chemical lysis (eg: sodium hydroxide), and / or by enzymatic lysis (eg: proteinase K). In a particular embodiment, the release of nucleic acids comprises a step of (i) lysis of the biological sample by a protease, in particular a serine protease, such as proteinase K, trypsine or chymotrypsine, more preferably proteinase K, followed by (ii) inactivation of the protease, either by temperature or preferably by an inhibitor of the protease such as phenylmethylsulfonyle fluorure (PMSF).

[0066] By ‘extracted from’, it means that the nucleic acids are released and purified from the biological sample, meaning that the nucleic acids are substantially purified from other components, e.g., RNA or DNA, or proteins, which naturally accompany the nucleic acid in the cell. When the target nucleic acid is a pathogen nucleic acid susceptible to have infected a subject or an environment sample, such nucleic acid template may be prepared by a mere lysis of the bacterial cell wall or the viral envelope by any method known in the art. For example, nucleic acids can be extracted using liquid extraction (e.g., Trizol, DNAzol) techniques, or using commercially available kits (e.g., ID Gene™ Mag Universal Extraction Kit, ID Gene™ Mag Fast Extraction Kit, NucleoSpin RNA Virus, QIAamp Circulating Nucleic Acid Kit, Qiagen DNeasy kit, QIAamp kit, Qiagen Midi kit, QIAprep spin kit). Nucleic acids can be concentrated or precipitated by known methods, including, by way of example only, centrifugation. Nucleic acids can be bound to a selective membrane (e.g., silica) for the purposes of purification. The nucleic acid extraction typically yields a solution comprising the isolated nucleic acid and a buffer, and optionally salts. This solution, or partition thereof, is then used in a step to amplify the desired target sequences.

[0067] In a particular and preferred embodiment, the invention is an in vitro method for detecting at least 3 target nucleic acid sequences, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 target nucleic acid sequences, preferably simultaneously, in the same reaction mix. The target nucleic acid sequences may be different sequences of a subject of interest that present genetic variations correlated to a physiological or pathological disorder or nucleic acids of one or several strains or pathogens that may have infected a subject (animal, human, plant) or a sample of environment.

[0068] Primers and tag sequence

[0069] Primers

[0070] As used herein, a “primer” refers to a single-stranded oligonucleotide or DNA fragment which hybridises with a nucleic acid strand (here, the target sequence) in such a manner that the 3' terminus of the primer can act as a site of polymerisation and extension using a DNA polymerase enzyme. A primer may have any suitable length, such as at least about 10, 15, 20, or 30 nucleotides. Preferably, the primer length is comprised between 15 and 30 nucleotides. In a preferred embodiment, the primers of the invention are produced synthetically. In some embodiments, a primer can be paired with a compatible primer within an amplification or synthesis reaction to form a primer pair consisting or a forward primer and a reverse primer. “Primer pair” thus refers to two primers comprising a forward primer that hybridises to a single strand at one end of the DNA sequence to be amplified (‘target sequence’), and a reverse primer that hybridises with the other end on the complementary strand of the DNA sequence to be amplified. Optionally, the forward primer primes synthesis of a first nucleic acid strand, and the reverse primer primes synthesis of a second nucleic acid strand, wherein the first and second strands are substantially complementary to each other, or can hybridise to form a double-stranded nucleic acid molecule. In some embodiments, one end of an amplification or synthesis product is defined by the forward primer and the other end of the amplification or synthesis product is defined by the reverse primer. Accordingly, a pair of primers may be a sense primer and an antisense primer that collectively define the opposing ends (and thus the length) of a resulting amplicon.

[0071] Typically, a primer is capable of hybridising to a corresponding target sequence and undergoing primer extension when exposed to amplification conditions in the presence of dNTPS and a polymerase. The term “hybridizing” refers to the formation of a doublestranded nucleic acid from complementary single stranded nucleic acids. The hybridization may occur between two nucleic acid strands perfectly matched or substantially matched with some mismatches. The complementarity for hybridization may depend on hybridization conditions, particularly temperature. In the rest of the description, we will use interchangeably the terms “primer” or “targetspecific primer” which refers generally to an oligonucleotide, that includes at least one sequence that is at least 50% complementary, typically at least 75% complementary or at least 85% complementary, more typically at least 90% complementary, more typically at least 95% complementary, more typically at least 98% or at least 99% complementary, or identical, to at least a portion of a nucleic acid molecule that includes a target sequence. In such instances, the target-specific primer and target sequence are described as “corresponding” to each other. In some embodiments, the target-specific primer is substantially non-complementary to other target sequences present in the sample. In some embodiments, the target-specific primer can include minimal cross hybridisation to other target-specific primers in the amplification reaction. In some embodiments, target-specific primers include minimal cross-hybridisation to non-specific sequences in the amplification reaction mixture. In some embodiments, a forward target-specific primer and a reverse target-specific primer define a target-specific primer pair that can be used to amplify the target sequence via template-dependent primer extension. Preferably, the target-specific primers include minimal self-complementarity.

[0072] Primers for amplification can have a concentration ranging from 0.01 pM to 1.0 pM, in particular from 0.02 pM to 1pM. Primers can be designed according to known parameters for avoiding secondary structures and self-hybridisation. Different primer pairs can anneal and melt at about the same temperatures, for example, within about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 °C of another primer pair. Primers can be prepared by a variety of methods including but not limited to cloning of appropriate sequences and direct chemical synthesis using methods well known in the art, or can also be obtained from commercial sources.

[0073] The primers can have an identical melting temperature. The melting temperature of a primer can be about 30 to about 85°C, about 40 to about 85°C, about 50 to about 85°C, or about 50 to about 65°C.

[0074] Tag sequence

[0075] A ‘tag sequence’ disclosed herein is a single strand oligonucleotide comprising a nucleotide sequence non-complementary to the target nucleic acid sequence, the said tag sequence being linked to the 5’-end portion of at least one primer of the primer pairs designed for a target nucleic acid. In the description, the expression ‘tag sequence’ and ‘tag sequence of the primer’ are used interchangeably. In a particular embodiment, the tag is placed upstream of the target part of the forward primer only, upstream of the target part of the reverse primer only or upstream of both primers at the same time. In a particular and preferred embodiment, the tag is placed upstream of one of the target primers.

[0076] By ‘tag primer’ or ‘primer-tag’ it means a primer comprising at its 5’-end portion, a tag sequence.

[0077] By ‘tag-free primer’, it means a primer that is free at its 5’-end portion, meaning it does not comprise a tag sequence.

[0078] In a particular embodiment, the concentration used in the mix reaction, of the ‘tag primer’ will be lower than the concentration of the ‘tag-free primer’. As an example, the reaction mix will comprise : the tag-free primer(s), at a concentration ranging from 0.05 to 0.3 pM, preferably 0.1- 0.2 pM, and the tag primer(s), at a concentration lower than the concentration of tag-free primer(s), in particular ranging from 0,01 pM to 0,05 pM, preferably 0.025 pM.

[0079] The tag sequence of the primer is designed so that its complementary sequence neosynthetized by the polymerase will hybridize with 3’ end portion of the probe sequence disclosed hereunder. The tag sequence of the primer is generally identical to the 3’ end portion of the probe, but the tag sequence and the 3’end portion of the probe may have some minor modifications, as long as it does not prevent the hybridization between the 3’ end portion of the probe and the complementary sequence of the tag sequence as neosynthetized by the polymerase.

[0080] In a particular embodiment, the tag sequence of the primer is the same sequence that the 3’ end portion of the probe sequence.

[0081] Each specific tag sequence of the primer corresponds generally to a probe sequence and is designed to detect a target nucleic acid.

[0082] In a particular embodiment, we will use the same tag and the same probe for several target nucleic acids, in particular several target nucleic acids of different strains of the same pathogen, in order to detect them with a unique signal (same melting curve).

[0083] In another embodiment, we will use different tags and different probes for several variants (mutations) of the same target nucleic acid sequence in order to detect them with differential signals. In another embodiment, we will use different tags and different probes for a same target nucleic acid, in order to detect them with a unique signal (same melting curve) and improve the sensitivity of the method. The tag sequence can be linked to the 5’-end portion of the primer according to well-known technologies.

[0084] In a preferred embodiment, the length of the tag sequence linked to the 5’end portion of at least one primer of the primers pair, ranges from 8 to 30 nucleotides, preferably from 10 to 20 nucleotides.

[0085] In the present invention, the melting temperature of a tag sequence generally ranges from 50°C to 65°C, preferably from 55°C to 62°C.

[0086] In a particular embodiment, the tag sequence of the primer has a linear structure with no interactive label (the interactive label is in the probe). In this embodiment, the corresponding probe sequence having an interactive label will hybridize with the 3’end portion of the extended neosynthetized single-strand, and will form, under conditions for inducing the 3’- extension reaction, an extended duplex providing the signal.

[0087] In a preferred embodiment, the length of the corresponding probe sequence disclosed hereunder ranges from 20 to 100 nucleotides, in particular from 30 to 60 nucleotides.

[0088] Embodiments for improving sensitivity and specificity of the detection

[0089] By ‘sensitivity’ of the method, it means the ability of the method to identify the target nucleic sequences in very small quantities, in other words the more the method is able to detect a small quantity, the more sensitive it is.

[0090] By ‘specificity’ of the method, it means the ability of the method to correctly identify the target nucleic sequences, in other words the method is able to only detect the target nucleic acid sequences (meaning it does not detect the other sequences).

[0091] In order to improve the sensitivity and specificity of the detection, man can use particular embodiments including (i) particular combinations of different tag sequences and probes with close melting temperature (Tm) ((Tm of the sequence region that will hybridize with the neosynthesized strand)), and / or (ii) particular conformation and / or composition of the tag sequence of the primer and / or of the probe sequence (melting probe).

[0092] Different tag sequences Indeed, and as mentioned above, a particular embodiment to increase the sensitivity of the method is to use 2 different tag sequences (1 on reverse primer and 1 on forward primer) and 2 corresponding probes. Indeed, the use of the same tag sequence to both reverse and forward primer will impact the amplification reaction because of circularisation of the sequence, with a loss of sensitivity.

[0093] Another particular embodiment to increase the sensitivity of the method is to use 2 different tag sequences (1 on reverse primer and 1 on forward primer) and 2 corresponding probes with close melting temperature (Tm), in order to obtain a unique signal (single peak).

[0094] Modifications of the primer-tag and the probe to decrease competition effect

[0095] Another way to improve the sensitivity and specificity of the detection is to decrease the competition between the melting probe, the primer tag and the complementary strand of the neosynthesized strand (to favorize hybridization of the neosynthesized strand on the melting probe), in particular by using modifications on the conformation and / or the composition of the primer tag sequence and the melting probe sequence.

[0096] In a particular embodiment, the tag sequence of the primer has a hairpin structure which allow a better sensitivity and specificity. Indeed, by folding over itself, the hairpin allows the restriction of the amplification outputs at low temperatures. The length of the hairpin sequence which bind to itself may range from 2 to 20 nucleotides, in particular from 3 to 10 nucleotides.

[0097] In another particular embodiment, 5’-Primer-tag modifications are also interesting to increase the melting temperature of the sequence and then optimize the sensitivity and specificity of the detection. Indeed, higher the melting temperature is, better is the binding on the target sequence and more specific is the reaction. As examples of 5’-Primer-tag modifications, mention may be made of 5’-Phosphate such as 5’-Thiophosphate or Triphosphates, 5’-Amino groups such as methyled amino groups (C3-C6-C12 Spacer for example), or 5’-Biotin. In a particular embodiment, the 5’-Primer-tag comprises a 5’-Phosphate such as 5’- Thiophosphate or Triphosphates.

[0098] Another embodiment to increase the melting temperature of the primer-tag sequence to promote the binding of the complementary neosynthesized strand with the 3’ end portion of the probe and then increase the sensitivity and specificity of the detection, is to use LNA or LNA like modified nucleotides on the primer-tag sequence. The number of LNA modification may range from 1 to 20 nucleotides. In another particular embodiment, the probe sequence has a hairpin structure which allow a better specificity of the detection. Indeed, by folding over itself, the hairpin allows the restriction of the amplification outputs at low temperatures. It also led to decrease the fluorescence background level to optimise the final intensity with the presence of a target. The length of the hairpin sequence which bind to itself may range from 3 to 30 nucleotides, in particular from 5 to 20 nucleotides.

[0099] Another embodiment to increase the melting temperature of the 3’ end portion of the probe sequence to promote the binding of the complementary neosynthesized strand with the 3’ end portion of the probe, and then increase the sensitivity and specificity of the detection, is to use LNA or LNA like modified nucleotides on the 3’ end portion of the probe sequence. The number of LNA modification may range from 1 to 20 nucleotides.

[0100] So, in a particular embodiment, the in vitro method of the invention comprise the primers-tag and / or the probes which have modifications in their conformation and / or their composition reducing the competition between primer-tag, probe and complementary strand of the neosynthesized strand and / or increasing the melting temperature of the 3’ end portion of the probe sequence to promote the binding of the complementary neosynthesized strand with the 3’ end portion of the probe, in particular selected in the group consisting of a primer-tag and / or probe having a hairpin structure, a 5’-Primer-tag modification including 5’-Phosphate, 5’-Amino groups, or 5’-Biotin, and / or a primer-tag and / or probe having LNA modification.

[0101] Amplification reaction and Melting analysis

[0102] Amplification reaction (PCR)

[0103] As used herein, “amplify”, “amplifying” or “amplification reaction” and their derivatives, refer generally to any action or process whereby at least a portion of a nucleic acid molecule (referred to as a template nucleic acid molecule) is replicated or copied through a templatedependent in vitro enzyme-catalysed reaction into at least one additional nucleic acid molecule. The template nucleic acid molecule can be single-stranded or double-stranded and the additional nucleic acid molecule can independently be single-stranded or doublestranded. Amplification optionally includes linear or exponential replication of a nucleic acid molecule.

[0104] One method which is commonly used for amplifying genetic material is the polymerase chain reaction (PCR) well known in the art. In PCR, nucleic acid primers that are complementary to opposite strands of a nucleic acid amplification target sequence are permitted to anneal to the denatured sample. Next, DNA polymerase (typically heat stable) extends the DNA duplex (forming an extended neosynthetized strand) from the hybridised primer. The process is then repeated to amplify the nucleic acid target. If the nucleic acid primers do not hybridise to the sample, then there is no corresponding amplified PCR product. In this case, the PCR primer acts as a hybridisation probe. Preferably, the PCR technique used quantitatively measures starting amounts of DNA, cDNA, or RNA. Examples of PCR-based techniques include techniques such as, but not limited to, quantitative PCR (Q-PCR), reverse-transcriptase polymerase chain reaction (RT-PCR), quantitative reversetranscriptase PCR (QRT-PCR), or digital PCR (dPCR). These techniques are well known and easily available technologies for those skilled in the art and do not need a precise description.

[0105] PCR is most usually carried out as an automated process with a thermostable enzyme. In this process, the temperature of the reaction mixture is cycled through a denaturing region, a primer annealing region, and an extension reaction region automatically. Machines specifically adapted for this purpose are commercially available. Improved and more sensitive PCR methods such as real-time PCR are preferred embodiments of the present invention.

[0106] In some embodiments, preparation of the nucleic acid solution may include combining the nucleic acid solution with reagents for amplification and for reporting whether or not amplification occurred (step of detection). Reagents for amplification may include any combination of primers for the targets, dNTPs and / or NTPs, at least one enzyme (e.g., a polymerase, a ligase, a reverse transcriptase, a restriction enzyme, or a combination thereof, each of which may or may not be heat-stable), and / or the like. Accordingly, preparation of the nucleic acid may render the nucleic acid solution capable of amplification of each of one or more targets, if present, in the nucleic acid solution.

[0107] Polymerase and reverse transcriptase

[0108] The polymerase can be a DNA polymerase. Examples of polymerases which can be used in the present method include a DNA Polymerase, a T4 DNA Polymerase, a T7 DNA Polymerase, a Taq DNA Polymerase, a Klen Taq Polymerase and any combination thereof.

[0109] Preferably, the DNA polymerase is a thermostable DNA polymerase, such as Taq DNA polymerase. When the target nucleic acid to be detected is an RNA, a reverse transcriptase may advantageously be added to the reaction mix in order to obtain a cDNA from the mRNA contained in the biological sample. The cDNA obtained will then be used as a target for amplification for the primers used in the method described herein.

[0110] Amplification conditions

[0111] Performing a reaction may include subjecting the reaction mix containing the target nucleic acids to one or more conditions that promote occurrence of the reaction. In some cases, amplification in the reaction mix may be encouraged by heating and / or incubating the reaction mix at a temperature above room temperature, such as at a denaturation temperature, an annealing temperature, and / or an extension temperature, for one or a plurality of cycles.

[0112] As used herein, the expression “amplification conditions” and its derivatives, generally refers to conditions suitable for amplifying one or more nucleic acid sequences. Such amplification can be linear or exponential. In some embodiments, the conditions suitable for amplifying one or more nucleic acid sequences includes polymerase chain reaction (PCR) conditions. Typically, the amplification conditions refer to a reaction mixture that is sufficient to amplify nucleic acids such as one or more target sequences. Generally, the amplification conditions include a catalyst for amplification or for nucleic acid synthesis, for example a polymerase; a primer that possesses some degree of complementarity to the nucleic acid to be amplified; and nucleotides, such as deoxyribonucleotide triphosphates (dNTPs) to promote extension of the primer once hybridised to the nucleic acid. The amplification conditions can require hybridisation or annealing of a primer to a nucleic acid, extension of the primer and a denaturing step in which the extended primer is separated from the nucleic acid sequence undergoing amplification. Typically, amplification conditions can include thermocycling; in some embodiments, amplification conditions include a plurality of cycles where the steps of annealing, extending and separating are repeated. Typically, the amplification conditions include cations such as Mg++or Mn++(e.g., MgCh, etc.) and can also include various modifiers of ionic strength.

[0113] The extension of the neosynthetized strand may be adjusted appropriately in consideration of the optimum temperature for the activity of the nucleic acid polymerase and the like.

[0114] In particular, the steps constituting the PCR cycle correspond respectively to step of : denaturation of the DNA (from 90° C. to 99° C.) , hybridization of the DNA to the primers and of elongation of the DNA by DNA polymerase starting from the primers (from 58° C. to 72° C.).

[0115] The cycle is typically repeated from 40 to 50 times, preferably 45 times.

[0116] Said PCR reaction is preferably preceded by a step of initial denaturation of the DNA contained in said biological sample, preferably at 95° C. for 3 or 10 minutes.

[0117] This step of initial denaturation is a heating step carried out before the PCR cycle. It makes it possible to prepare the DNA of the sample, which will serve as a matrix during the amplification reaction, in particular by completely dehybridizing the double-stranded DNA, by disrupting the secondary structures of the DNA or also by activating the DNA polymerase.

[0118] Said PCR reaction, as illustrated in the examples, can be carried out using a reaction mixture comprising at least: the DNAs and / or RNAs target sequences contained in the biological sample, the tag-free primer(s), at a concentration ranging from 0.05 to 0.15 pM, preferably 0.1 pM the tag primer(s), at a concentration lower than the concentration of tag-free primer(s), in particular ranging from 0,01 pM to 0,05 pM, preferably 0.025 pM, the corresponding probe(s) at a concentration ranging from 0,02 pM to 0,05 pM, preferably 0.033 pM, and at least one unit of DNA polymerase and optionally Reverse transcriptase (in case of RNAs target sequences).

[0119] Melting

[0120] In a particular embodiment, the PCR reaction is a qPCR coupled with melting analysis.

[0121] In this embodiment, the molecular biology technique is carried out starting from doublestranded DNA target sequences that have been amplified by PCR as disclosed above. The sample then contains a large number of copies of the DNA fragments targeted and amplified by the PCR.

[0122] The melting analysis then consists of precise, controlled heating of the DNA target fragments amplified by PCR to cause their denaturation. Monitoring the denaturation of the DNAs, during the melting analysis, thus makes it possible to determine a specific melting profile of each target DNA fragment.

[0123] The “melting profile” (also called “melting curve”) corresponds to the development of the denaturation of one (or on average of each) DNA molecule as a function of the temperature. Within the meaning of the invention, a melting curve is not necessarily a graphical curve, but can be a list or a table of values of several points of this curve during this denaturation of one (or on average of each) DNA molecule as a function of the temperature.

[0124] Thus, the method according to the invention comprises acquisition of at least one normalized curve (as seen above) of DNA melting of the measurement biological sample, called at least one measurement curve, each measurement curve comprising different points, each point corresponding to a quantity that is proportional to (for example a fluorescence signal) or representative of a level (typically in %) or of a quantity of denaturation of the DNA of the measurement sample as a function of a temperature; this acquisition can comprise carrying out the PCR and the melting curve itself (in the laboratory), and / or a simple downloading of data (computer data for example) of this melting curve.

[0125] In a particular and preferred embodiment, said PCR reaction is followed by a step of gradual heating between 60° C. and 100° C., preferably from 65° C. to 97° C., in particular 97°C to produce denaturation of said amplification products, and obtain a melting profile of each amplification product.

[0126] After amplification, the melting programme is carried out using a thermocycler. This step of gradual heating corresponds to heating of the sample carried out in a controlled manner, during which the temperature gradually increases in stages over time, following the suppliers’ recommendations for a thermocycler. As illustrative but non-limitating examples, for Mic qPCR cycler, we use an increase of 0,2°C / s to 0,5°C / s, in particular 0,35°C / s ; for QuantStudio™ 5 / 7 cycler, we use an increase of 0,02°C / s to 0,1°C / s, in particular 0,05°C / s ; and for CFX96 cycler, we use an increase of 0,1°C / cycle to 0,5°C / cycle, in particular 0,2°C / cycle.

[0127] In a particular embodiment and as illustrated in the Examples, the steps of amplification and melting were carried out using specific primers pairs (forward, reverse, forward-tag, reversetag primers) and Melting Probes (MP) designed for detecting at least two target nucleic acids, in a reaction mix comprising a DNA polymerase (and optionally a Reverse transcriptase) and the essential components for amplification of the targets and melting step. The amplification procedure consists of an initial denaturation followed by 45 cycles of denaturation, hybridization and elongation. After amplification, the melting programme is carried out using a thermocycler, such as a Mic qPCR Cycler (Bio Molecular Systems) for example, by heating to 95° C. for 1 minute, cooling to 40° C. for 1 min, followed by the application of a temperature increase from 65 to 97° C. at a rate of increase of 0.35° C. / s and continuous measurement of the fluorescence. The obtained amplification curves and melting profiles are associated with the detection of each targeted nucleic acid sequence.

[0128] Advantageously, the PCR for this melting curve is carried out in the simultaneous presence of several primer pairs targeting several target DNA molecules. We then refer to “multiplexing” conditions.

[0129] Multiplex amplification and melting analysis

[0130] In the method of the invention, the amplification step is a multiplex PCR amplification that includes the simultaneous amplification of at least two target sequences in a single amplification reaction. Thus, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25 sequences are amplified, preferably simultaneously amplified, in step b) of the present method.

[0131] The technology of the invention is then called Multiplex Probe Melting or“MPM technology”.

[0132] The several target sequences can be different target nucleic acid molecules included in the single amplification reaction or different sequences (e.g mutations) localized on the same nucleic acid molecule.

[0133] Therefore, a plurality of target sequences is amplified by a single PCR using a primer pool for amplifying each target sequence. Multiplex PCR assays are well known in the art. See e.g., WO 2010 / 115100.

[0134] A set of primers for a multiplex PCR should be capable of specifically binding to a target sequence and should not interfere with each other in order to amplify the target sequence by a sufficient amount. A multiplex PCR using such a set of primers saves time, effort and cost for amplifying a target sequence in comparison with a single PCR. Moreover, multiplex PCR lowers the threshold of detection of nucleic acid in a sample. In particular, even fragments of the target nucleic acid can be detected when several pairs of primers targeting different sequences are used. In other words, multiplex PCR enables the detection of target sequences even when the starting material is degraded, thereby increasing the sensitivity of the assay.

[0135] In order to multiplex and discriminate several targets in a single reaction mix, it is essential to differentiate the detection signals with different Probes (also named Melting Probes or ‘MP’ in the Examples). In particular, a distance between quencher and reporter on the probe and optionally a distance between 3’ blocker and reporter is (are) specified for each target sequence to be detected. The distinct melting temperature signals are obtained by increasing the distance between the reporter and the quencher, as disclosed hereunder in the ‘Probe and detection’ chapter and illustrated in the Examples. The melting temperature (Tm) also depends on the composition of the sequence (%GC).

[0136] In a Multiplex system, PCR amplification for each multiplex can be performed using the same thermal cycling profile thereby allowing the amplification of all the nucleic acid targets at the same time (simultaneously) in a single apparatus (e.g., thermocycler).

[0137] In a particular embodiment, the PCR reaction is a multiplex PCR reaction comprising at least two primer pairs targeting at least two target nucleic acids and at least one tag sequence per primer pair, for amplifying, detecting and optionally quantifying simultaneously, at least two target nucleic acids, preferably a multiplex PCR reaction coupled with melting analysis.

[0138] In another particular embodiment, man can use one tag and one probe targeting several nucleic acid sequences of a same pathogen (eg variants or strains for a pathogen) to provide a unique signal (same melting profile), as illustrated in the Example 2.

[0139] In a particular embodiment, the multiplex PCR reaction is carried out with 2 to 25 target nucleic acids, in particular 2 to 10 target nucleic acids, either in a sole channel or in several channels.

[0140] So, one of the advantages of the method of the invention is to allow the multiplex detection of several targets from the same reaction mix, and even simultaneously in some cases. Figure 6 illustrates that it is possible to obtain signals from a reaction mix targeting simultaneously two infectious agents. Indeed, both melting profiles are observed as expected at temperatures of 79.7°C for Metapneumovirus and 82.8°C for Rhinovirus. These results demonstrated that it is possible to develop the method of the invention with the objective to add more targets and multiply them to detect more targets in the same reaction mix. The method of the invention is also capable of correctly discriminating the fusion profiles of two targets simultaneously. Then, it is possible to extend this method to a reaction mix containing more than two targets. Indeed, the method of the invention is particularly suitable for the detection of multiple targets, even within the same well.

[0141] Controls

[0142] In some embodiments, the PCR comprises amplifying not only sequences of the target nucleic acids of interest, but also at least one sequence of an unrelated nucleic acid, said sequence being an endogenous sequence (internal control) or an exogenous sequence (external control). Accordingly, an endogenous sequence that is known or expected to be present in sample (internal control), and / or that has a known or expected representation with respect to a bulk nucleic acid population present in the sample (e.g., total DNA, total genomic DNA, genomic DNA from a particular species of organism, total RNA, total mRNA, etc.), may be amplified in the multiplex PCR assay. In contrast, target reagents may amplify an exogenous sequence that has an unknown presence in the sample and / or an unknown presence in with respect to the bulk nucleic acid population (external control). In any event, amplification of the control sequence (internal or external control) may be used to determine the quality of test data measured from a sample, such as to verify the quantitative accuracy of the test data and / or to determine the reliability of the test data.

[0143] Control sequence reagents may be similar in general structure to the test target reagents, but different with respect to the nucleic acid sequences of the primers and probes, to provide test target and control target specificity, respectively. Also, the test and control probes may include distinct dyes and / or distinct energy transfer partners (e.g., distinct quenchers suitable for the respective dyes).

[0144] The control sequence may be a positive control of sample preparation, nucleic acid extraction, and amplification. Furthermore, an amount of control sequence determined to be present in the sample may provide a standard against which an amount of test target determined to be present in the sample can be compared and / or normalised.

[0145] Probes and Detection of the signal

[0146] Reagents for reporting (or detection) may include a different reporter (probe) for each target sequence of interest. Accordingly, preparation of the nucleic acid solution for reporting may render the nucleic acid solution capable of reporting, or being analysed for, whether or not amplification has occurred, on a target-by-target basis, and optionally the extent of any such amplification. The reporters each may be a labelled probe that includes a nucleic acid (e.g., an oligonucleotide) labelled with a luminophore (i.e. , a photoluminescent moiety), such as a fluorophore.

[0147] A “probe” as used herein refers to a nucleic acid connected to at least one label. As used herein, a “label” is an identifying and / or distinguishing marker or identifier connected to or incorporated into any entity. A label may, for example, be a dye that renders an entity optically detectable and / or optically distinguishable. Exemplary dyes used for labelling are fluorescent dyes (fluorophores) and fluorescence quenchers. In the present invention, a probe sequence hybridizes with the extended neosynthetized single-strand and forms, under conditions for inducing the 3’-extension reaction, an extended duplex providing the signal. Indeed, the probe sequence comprises, at its 3’ end portion, a sequence which is identical (or with minor modifications) to the tag sequence of the primertag, in order to hybridize with the extended neosynthetized single-strand and forms, under conditions for inducing the 3’-extension reaction, an extended duplex providing the signal.

[0148] The term “signal” in the present invention means any signal capable of indicating the presence or absence of the extended duplex. The detectable signal is detected during or after the amplification reaction. The detection of the signal indicates the presence of the extended duplex, and the presence of the extended duplex indicates amplification (extension) of the extended duplex. For example, the signal includes a signal from labels (signal generation or extinguishment), a signal change from labels (signal increase or decrease), a melting curve, a melting pattern and a melting temperature (or Tm value).

[0149] The probe(s) used may thus comprise a component comprising at least one detectable label. The ‘detectable label’ may be capable of producing an optical signal. Alternatively, the detectable label may comprise a fluorophore. Examples of fluorophores include but are not limited to fluorescent proteins, for example GFP (green fluorescent protein), YFP (yellow fluorescent protein), RFP (red fluorescent protein); non-protein fluorophores selected from the group consisting of xanthene derivatives (for example, fluorescein, rhodamine, Oregon green, eosin, 6-carboxyfluorescein and Texas red); cyanine derivatives (for example, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine), squaraine derivatives and ring-substituted squaraines, including Seta, SeTau, and Square dyes, naphthalene derivatives (dansyl and prodan derivatives), coumarin derivatives, oxadiazole derivatives (for example pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole), anthracene derivatives (for example anthraquinones, DRAQ5, DRAQ7 and CyTRAK Orange), pyrene derivatives(for example cascade blue), oxazine derivatives (for example, Nile red, Nile blue, cresyl violet, oxazine 170), acridine derivatives (for example proflavin, acridine orange, acridine yellow), arylmethine derivatives (for example auramine, crystal violet, malachite green), tetrapyrrole derivatives (for example porphin, phthalocyanine, bilirubin) and derivatives thereof. As examples of reporters and quencher molecules, mention may be made of : Cy2™ (506), YO-PRO™-1 (509), YOYO™-1 (509), Calcein (517), FIT (518), FluorX™ (519), Alexa™ (520), Rhodamine 110 (520), Oregon Green™ 500 (522), Oregon Green™ 488 (524), RiboGreen™ (525), Rhodamine Green™ (527), Rhodamine 123 (529), Magnesium Green™ (531), Calcium Green™ (533), TO-PRO™-1 (533), TOTO1 (533), JOE (548), BODIPY530 / 550 (550), Dil (565), BODIPY TMR (568), BODIPY558 / 568 (568), BODIPY564 / 570 (570), Cy3™ (570), Alexa™ 546 (570), TRITC (572), Magnesium Orange™ (575), Phycoerythrin R&B (575), Rhodamine Phalloidin (575), Calcium Orange™ (576), Pyronin Y (580), Rhodamine B (580), TAMRA (582), Rhodamine Red™ (590), Cy3.5™ (596), ROX (608), Calcium Crimson™ (615), Alexa™ 594 (615), Texas Red(615), Nile Red (628), YO-PRO™-3 (631), YOYO™-3 (631), R-phycocyanin (642), C-Phycocyanin (648), TO-PRO™-3 (660), TOTO3 (660), DiD DilC (5) (665), Cy5™ (670), Thiadicarbocyanine (671), Cy5.5 (694), HEX (556), TET (536), Biosearch Blue (447), CAL Fluor Gold 540 (544), CAL Fluor Orange 560 (559), CAL Fluor Red 590 (591), CAL Fluor Red 610 (610), CAL Fluor Red 635 (637), FAM (520), Fluorescein (520), Fluorescein-C3 (520), Pulsar 650 (566), Quasar 570 (667), Quasar 670 (705) and Quasar 705 (610), wherein the numeric in parenthesis is a maximum emission wavelength in nanometer. Preferably, the fluorophore is selected from the group consisting of FAM (carboxyfluorescein), TET (carboxy-2',4,7,71tetrachlorofluorescein succinimidyl ester), HEX (carboxy-2,4,4,5,7,7 - hexachlorofluorescein succinimidyl ester), ROX (carboxy-X-rhodamine) and NED. More preferably, the fluorophore is FAM (carboxyfluorescein) or 6-FAM (6-carboxyfluorescein), or HEX (carboxy-2,4,4,5,7,7 -hexachlorofluorescein succinimidyl ester).

[0150] In a particular embodiment, a non-fluorescent black quencher molecule capable of quenching a fluorescence of a wide range of wavelengths or a specific wavelength such as BHQ and DABCYL, is used in the present invention.

[0151] Interactive pair of labels (fluorophore and quencher) on the probe

[0152] The probe sequence (also named Melting Probe (MP) in the technology of the invention) comprises interactive label system including a reporter molecule and a quencher molecule.

[0153] As a representative of the interactive label system, the FRET (fluorescence resonance energy transfer) label system includes a fluorescent reporter molecule (donor molecule) and a quencher molecule (acceptor molecule). In a particular embodiment, the label system includes a fluorescent reporter molecule and two quenchers molecules (one of the quenchers can be the 3’ blocker).

[0154] Before the extended duplex is formed, i.e. , before the probe hybridizes with the 3’-end portion of the neosynthetized single-strand, the fluorescent reporter molecule and the quencher molecule are conformationally adjacent to each other to allow the quencher molecule to quench the signal from the reporter molecule (‘no signal’).

[0155] In contrast, when the extended duplex is formed, the fluorescent reporter molecule and the quencher molecule are conformationally separated to allow the quencher molecule to unquench the signal from the reporter molecule, giving rise to changes in signals from the interactive dual label (‘providing signal’). Thus, such signal change indicates the formation and presence of the extended duplex, and the formation and presence of the extended duplex indicates amplification of target sequence.

[0156] In a particular embodiment, the probe sequence has a linear structure.

[0157] As disclosed above, in order to improve the sensitivity and specificity of the detection, man can use particular embodiments including particular conformation and / or composition of the tag sequence as disclosed above, and / or particular conformation and / or composition of the probe sequence.

[0158] In a particular embodiment, the probe sequence has a hairpin structure which allow a better sensitivity of the detection. Indeed, by folding over itself, the hairpin allows the restriction of the amplification outputs at low temperatures. It also led to decrease the fluorescence background level to optimise the final intensity with the presence of a target. The length of the hairpin sequence which bind to itself may range from 3 to 30 nucleotides, in particular from 5 to 20 nucleotides.

[0159] Another embodiment to increase the melting temperature of the 3’ end portion of the probe sequence to promote the binding of the complementary neosynthesized strand with the 3’ end portion of the probe to promote the binding and then increase the sensitivity and specificity of the detection, is to use LNA or LNA like modified nucleotides on the probe sequence. The number of LNA modification may range from 1 to 20 nucleotides.

[0160] Preferably, the interactive pair of labels may comprise a fluorophore and a quencher pair. In one specific aspect, the fluorophore may be located at the 3' end or in the middle of the probe, and the quencher may be located at the 5 'end of the probe. Examples of quenchers include but are not limited to TaqMan® MGB (minor groove binder), NFQ (Non Fluorescent Quencher), DABCYL, EDQ, QXL, MGB and BHQ™ (Black Hole Quencher™). In one specific example, the fluorophore is FAM (6-carboxyfluorescein), or HEX (carboxy-2,4,4,5,7,7 -hexachlorofluorescein succinimidyl ester also named hexachlorofluorescein), and the quencher is BHQ™ (Black Hole Quencher™).

[0161] The distance between the reporter molecule and the quencher molecule and / or the distance between the reporter molecule and the 3’ blocker (which may be itself the quencher) can be used to differentiate the signals for each target sequence. Indeed, and as disclosed above, in order to multiplex and discriminate several targets in a single reaction mix, it is essential to 1 differentiate the detection signals with different Probes (also named Melting Probes or ‘MP’ in the Examples), wherein a distance between quencher and reporter on the probe and / or a distance between the reporter molecule and the 3’ blocker is specified for each target sequence to be detected. The distinct melting temperature signals are obtained by increasing the distance between the reporter and the quencher. Besides, the fluorescence signal of the reporter is correctly inhibited by the quencher when these two components are separated by 15 to 55 nucleotides for example. The method of the invention allows a fluorescence signal to be obtained correctly, no matter the distance between the reporter and the quencher is 15 or 55 nucleotides.

[0162] The distance between the reporter molecule and the 3’ blocker may be defined and adjusted to obtain a differential signal. According to an embodiment of the present invention, the reporter molecule is located at 10 to 25 nucleotides apart from 3'-end of the probe and the quencher molecule is located to quench the signal from the reporter molecule. In a particular embodiment, the reporter molecule is located at 15 nucleotides apart from 3'-end of the probe.

[0163] In a particular embodiment, the distance between the fluorophore and the quencher on the probe sequence ranges from 5 to 70 nucleotides, in particular 10 to 60 nucleotides, preferably 15 to 55 nucleotides.

[0164] The probe(s) used in the method disclosed herein have a length of at least 20, at least 30, at least 40, or at least 50 nucleotides. Alternatively, the length of the probe(s) used in the present method is less than 100, less than 90, less than 80 nucleotides. So, in a particular embodiment, the length of the probe(s) sequence ranges from 20 to 100 nucleotides, in particular from 30 to 60 nucleotides.

[0165] In a particular embodiment, probes nucleic acid sequences may advantageously comprise locked nucleic acids (LNA) (modified nucleic acids), in order to increase the melting temperature (Tm) of probes (Tm of the sequence region that will hybridize with the neosynthesized strand), which will optimize their specificity to the targeted DNA sequence. Mention may be made to the publication of Matthew P. Johnson et al. (Nucleic Acids Research, 2004, Vol. 32, No. 6 e55DOI: 10.1093 / nar / gnh046) for the LNA technology. In addition and in order to avoid non-specific signals with the method of the invention, the 3' end of the probe used in the method of the invention must be blocked by a ‘3’ blocker’.

[0166] 3’ blocker on the probe

[0167] The term ‘3’ blocker’ in the present invention encompasses any molecule linked to the 3’-end portion of the probe sequence that will prevent a DNA polymerase from binding and / or that will block extension at the 3’-end of the probe. So only extension from the 3’-en portion of the neosynthetized strand will occur, forming the extended complex.

[0168] The molecule used as ‘blocker’ may be a high molecular molecule whose stoichiometry will prevent binding of the DNA polymerase, in particular prevent the binding of the Taq polymerase.

[0169] In particular, the 3’ blocker is selected in the group consisting of C3 spacer, Biotin C3, Biotin DR, quencher, phosphates, inverted nucleotide bases, blocked nucleotides (dC), or amine groups. So in a particular embodiment and as illustrated on Figure 4, the 3’ blocker is selected in the group consisting of 3’ Spacer C3, 3’ BHQ1, 3’ dC blocked, 3’ inverted base dC, 3’ Biotin C3 and 3’ Biotin dR.

[0170] It is also possible to use the quencher positioned at 5' directly as a blocker at 3'.

[0171] So, in a particular embodiment, the 3’ blocker is the quencher.

[0172] This allows the two actions necessary for the functionality of the method of the invention to be mutualised, as illustrated in Figure 5. This alternative allows the sensitivity and specificity of the method of the invention to be maintained, while reducing the number of modifications to the melting probe.

[0173] Different channels

[0174] It is also possible to perform multiplex target detection using different channels.

[0175] So in a particular and preferred embodiment, the multiplex PCR reaction is carried out with 2 to 25 target nucleic acids, in particular 2 to 10 target nucleic acids, in several channels.

[0176] As illustrated in the Examples, Figure 7 shows that it is possible to obtain amplification curves and melting profiles on the HEX channel for the detection of Meta pneumovirus. The melting profile obtained with the melting probe containing the HEX fluorophore on the HEX channel is very similar to the one obtained with the melting probe containing the FAM fluorophore on the FAM channel. This means that the method of the invention can be used with different other fluorophores. As a results, different targets can be detected using different fluorescence channels, so the method the invention allows the detection of a multitude of targets from several fluorescence channels.

[0177] QUANTIFICATION OF THE TARGET NUCLEIC ACIDS (OPTIONAL)

[0178] In a particular embodiment, the method of the invention also further comprises a step of c) quantifying the amount of the said target nucleic acids as amplified in step b). This step may comprise the use of a probe, such as a Taqman probe, specific to the target nucleic sequence and distinct from the probe hybridizing the tag sequence.

[0179] Such probe is labelled by a fluorophore or an interactive label as disclosed above.

[0180] Quantifying the target nucleic acid is advantageous since it allows easy comparison between different samples or different target nucleic acids in a single sample.

[0181] As illustrated in the Example 7 and Figure 8, it is possible to combine the technology of the invention with a real-time PCR detection using a TaqMan probe. Therefore, different targets can be detected in the same well using these two different technologies, some targets (e.g sequences of a pathogen) being detected by using the technology of the invention using a melting probe (MP) and some other targets (e.g sequences of another pathogen distinct from the previous one) being detected by a real-time PCR detection using a TaqMan probe. This allows to offer increased multiplexing, as well as the possibility to specifically quantify a target if required. Indeed, the analysis of the amplification curve obtained by the RT-PCR using the TaqMan probe will potentially provide a quantitative or qualitative information of the target sequence present in the biological sample.

[0182] AUTOMATIZED SYSTEM

[0183] In a particular embodiment, the qPCR coupled to melting analysis is automatized and coupled with a method for classification of target signals of a biological sample, as disclosed for example in the patent application US2019050535 comprising : acquisition of at least one DNA melting measurement curve of the biological sample, determination that the measurement biological sample belongs to a defined group among different possible groups, by analysis of descriptors originating from the at least one measurement curve, characterized in that the descriptors comprise one or more points of the first and / or second derivative of each measurement curve. In particular, such method comprises : acquisition of at least one DNA melting curve of the measurement biological sample, called at least one measurement curve (preferably acquisition of several DNA melting curves of the measurement biological sample, also called technical replicates), typically each measurement curve comprising different points, each point corresponding to a quantity that is proportional to or representative of a level or a quantity of denaturation of the DNA of the measurement sample as a function of a temperature, determination that the measurement biological sample belongs to a defined group among different possible groups, by analysis of descriptors originating from the at least one measurement curve, characterized in that the descriptors comprise: one or more (preferably several) point(s) of the first derivative of each measurement curve and / or comprise one or more (preferably several) point(s) of the second derivative of each measurement curve, and / or one or more (preferably several) point(s) of the first derivative of each measurement curve and comprise one or more (preferably several) point(s) of the second derivative of each measurement curve, and / or one or more point(s) of each measurement curve, and / or one or more percentile(s) of each measurement curve.

[0184] The acquisition of at least one DNA melting curve of the measurement biological sample can comprise the acquisition of at least one melting curve of a result of a PCR obtained in the simultaneous presence of several primer pairs targeting several target DNA molecules, corresponding for example to several pathogens. This is then referred to as “multiplexing” conditions. This embodiment is useful for speeding up the search, for example, for several pathogens that are very rarely present together in one and the same biological sample. The rare cases for example of several pathogens present together in a sample are identified: typically, the melting curve has as many inflection points as pathogens present; different “defined groups” can then include the different combinations of the presence of these different pathogens.

[0185] The determination can comprise determination by a random forest method. The method according to the invention can comprise learning comprising: acquisition of different DNA melting curves, called reference curves, starting from different reference biological samples belonging to different known initial groups determined before learning, then determination of the descriptors from the reference curves, and then construction of a forest by the random forest method, comprising construction of several trees by the random forest method, each tree comprising, at each node, the use of one of the descriptors associated with a threshold and making it possible to separate the reference curves into two subsets, each leaf of each tree only corresponding to a single group among the different possible groups.

[0186] Some embodiments and device for such automatized method are disclosed in the patent application LIS2019050535.

[0187] KIT

[0188] The present invention also relates to use of a kit in the in vitro method of the invention for detecting at least two target nucleic acid sequences from a biological sample, which comprises : a) a specific primers pair for each target nucleic acid sequence, wherein at least one primer of the pair additionally comprises a tag sequence that is linked to its 5’-end portion; b) a template-dependent nucleic acid polymerase capable of inducing the 3’-extension reaction at the 3’-end of the primer and forming an extended neosynthetized strand; c) at least one probe sequence comprising a 3’ blocker and an interactive label, wherein the probe sequence hybridizes with the 3’-end portion of the extended neosynthetized singlestrand ; d) optional reagents for denaturing the amplified products and performing target amplification PCR such as buffers, DNA polymerase cofactors, deoxyribonucleotide-5-triphosphates, and reverse transcriptase ; e) optional reagents necessary for performing positive and negative control reactions ; and f) optional reagents for detecting the extended duplex formed with the probe sequence comprising the interactive label.

[0189] APPLICATIONS As mentioned above, the method of the invention is advantageous for multiplex detection of several nucleic acids from a biological sample, preferably at the same time (simultaneously) and in a single apparatus (thermocycler). In this embodiment, the MPM technology (Multiplex Melting Probe) of the invention provide detection of at least 2 target nucleic acid sequences, in particular at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or at least 25 target nucleic acid sequences, preferably simultaneously, in the same reaction mix.

[0190] Depending on the number of target nucleic acids molecules to be detected in the same reaction mix, and as illustrated in the examples hereunder, the man skilled in the art will play with the different tools, even in combination, to multiply the number of differential signals (melting curves) necessary to detect several targets from a single biological sample or in the same reaction mix : one or several tags and related probes targeting the said nucleic acids (1 tag and probe per target nucleic sequence as illustrated in example 5 or 1 tag and probe per ‘pool’ of target nucleic sequences of the same family as illustrated in example 2); different lengths between the fluorophore and the quencher on the probe, as illustrated in example 3; and / or different fluorophores (1 fluorophore per target nucleic sequence or 1 per ‘pool’ of target nucleic sequences of the same family) as illustrated in example 6.

[0191] By ‘pool’ of target nucleic sequences of the same family, it means for example target nucleic sequences belonging to different strains of a same pathogen, or different variants of a same gene.

[0192] The method of multiplex detection of several nucleic acid sequences opens new ways to analyse, even simultaneously and in the same wall, from 2 to 25 target nucleic acids sequences.

[0193] The method of the invention can be used in many domains of molecular biology, such as the infectious diagnosis of several pathogens within the same sample, or to analyse mutations of interest within the same microbial strain in particular for monitoring the epidemiology of microbial outbreaks. The method of the invention can also be used to detect any type of molecular markers, for example oncology markers, inflammation markers, and many others in a biological sample of a subject of interest.

[0194] So, the present invention also concerns use of the in vitro method according the invention or use of the kit for detecting, and optionally quantifying, nucleotide variations or mutations, in particular variations or mutations correlated with genetic or phenotypic traits in a subject (for genotyping or screening applications) and / or correlated with physiological disorders of a subject (for detecting or monitoring a disorder). By "nucleotide variation" in the present invention, it encompasses any single or multiple nucleotide substitutions, deletions or insertions in a DNA sequence at a particular location among contiguous DNA segments that are otherwise similar in sequence. Such contiguous DNA segments include a gene or any other portion of a chromosome. These nucleotide variations may be mutant or polymorphic allele variations. For example, the nucleotide variation detected in the present invention includes SNP (single nucleotide polymorphism), mutation, deletion, insertion, substitution and translocation. In particular, another object of the invention is use of the in vitro method according to the invention or use of the kit according to the invention for (i) detecting and optionally quantifying nucleic acids target sequence(s) (eg genes or molecular markers) in a biological sample such as animal, human, plant, or environmental sample, and / or (ii) screening and selecting genes or markers of interest, notably in the domains of health, virology, agriculture, ecological or environmental field, in particular detecting and optionally quantifying, nucleotide variations or mutations in a subject, such as variations correlated with genetic or phenotypic traits of the subject , or variations or mutations correlated with physiological disorders of a subject, and / or detecting and optionally quantifying nucleic acids of pathogens having infected the subject or pathogens present in a sample of environment.

[0195] So the method of the invention can be used in particular for the diagnosis, prognosis or monitoring of the physiological state of a given subject of interest.

[0196] The method and kit of the invention can also be used to detect any type of molecular markers, for example oncology markers, inflammation markers, and many others in a biological sample of a subject of interest.

[0197] It can also be used in genotyping or screening project, to select the microorganism’s gene, or to select seeds in the agricultural field. In this field using the method of the invention could also allow the specific detection of phytopathogen organism.

[0198] In a particular embodiment, the in vitro method according to the invention or the kit according to the invention are used for detecting and / or quantifying nucleic acids of pathogens having infected the subject or pathogens present in a sample of environment.

[0199] So in a particular embodiment, the method of the invention can be used for the diagnosis, prognosis or monitoring of the physiological state of a given subject of interest, infected or susceptible to be infected by a pathogen or affected by a physiological or pathological disorder. Another application is the detection and / or monitoring of pathogens in a sample of environment or a sample of a population of subjects for monitoring the epidemiology of microbial outbreaks.

[0200] Hereinbelow, the present invention is explained in detail in view of the examples. However, the following examples are given only for exemplification of the present invention, and it is evident that the present invention is not limited to the following examples.

[0201] EXAMPLES

[0202] To evaluate the performance of the method of the invention, it has been carried out on different types of nucleic acids and on different bacterial or viral strains. As illustrative examples, results were observable on synthetic DNA (gBIocks™ IDT) or RNA corresponding to viral strains of respiratory pathogens (ATCC-Zeptometrix) or bacterial strains of pathogens causing bovine mastitis, in particular Streptococcus agalactiae such as disclosed in Example 1. This last model demonstrates the proof of concept of the method of the invention based on real-time PCR coupled with a melting step.

[0203] The sequences of the forward, reverse, forward-tag, reverse-tag primers and the Melting Probe (MP) used in the following examples are described in the Table 1 hereunder:

[0204] Table 1 : Oligonucleotides sequences uses in the examples

[0205] In the primers-tag listed hereunder, the characters in bold type correspond to the ‘tag sequence’ and respectively in the probe sequences listed hereunder, the characters in bold type correspond to the 3’ end portion of the probe sequence which will hybridize with the neosynthesized single-strand.

[0206] BHQ1= Black Hole Quencher 1

[0207] 6-FAM = 6-Carboxyfluorescein

[0208] HEX = Hexachlorofluorescein

[0209] BHQ2= Black Hole Quencher 2

[0210] Example 1 : Detection of DNA nucleic sequence by the method of the invention

[0211] The sequences of the forward, reverse, forward-tag, reverse-tag primers and the Melting Probe (MP) used in this example are described in Table 1. It is possible to use the method of the invention by positioning the tag upstream of the target part of the forward primer only, of the reverse primer only or upstream of both primers at the same time. Three reaction mixes were then performed to validate the feasibility of both tag positioning independently and simultaneously. Each reaction is composed of 0.1 pM of target primers without tag and 0.025 pM of target primers with tag. In the case where the tag is placed on both primers simultaneously, the concentration of tagged primers used is 0.025 pM. A concentration of 0.033 pM of MP corresponding to the detected target is also added to the reaction. Taq polymerase and the essential components for its action are added with Lyo-Ready 1-Step RT-qPCR Virus Master Mix (Meridian Bioscience™) according to the supplier's recommendations. An input volume of 5 pL of synthetic Streptococcus agalactiae DNA or water (negative control) are put in 15 pL of each reaction mix previously dispensed in a PCR plate.

[0212] The amplification of synthetic DNA by real-time PCR coupled with a melting step is performed on a Mic qPCR Cycler (Bio Molecular Systems) according to the amplification program shown in Table 2.

[0213] Table 2: Amplification program using real-time PCR coupled with a nucleic acid fusion step.

[0214] The results observed in Figure 1 show that it was possible to obtain amplification curves and melting profiles associated with the detection of S. agalactiae with the method of the invention. When water (negative control) is added to the reaction mix, neither amplification curves nor melting profiles are obtained, which validates the specificity of the profiles on the synthetic target DNA.

[0215] Furthermore, it was confirmed that the positioning of the tag on the sense or antisense primer does not modify the obtained curves, but according to the conditions used in the present example, the results show that it is preferable to position the tag upstream of one of the target primers. So as shown in Figure 1 , the invention described herein can be implemented in several ways.

[0216] Example 2: Comparison of the method of the invention (MPM technology) with a classical high-resolution DNA melting (HRM)

[0217] In a second step, it was necessary to ensure that the obtained melt profile corresponds to a unique profile controlled by the MP composition, independently to the genetic variations of a pathogen. Therefore, a comparison between a classic HRM reaction mix and a MPM reaction mix according to the invention was performed. Table 1 shows the different sequences used to obtain the results shown in Figure 2.

[0218] The first mix (Classic HRM mix) contained the Influenza B specific primers at 0.1 pM each, an intercalating dye, the LightCycler® 480 High Resolution Melting Dye (Roche) to be used according to the supplier's recommendations, and the Master Mix Lyo-Ready 1-Step RT- qPCR Virus (Meridian Bioscience™) with its Reverse Transcriptase to be used according to the supplier's recommendations too. The second mix (MPM mix) is composed of 0.1 pM of tag-free forward primers targeting an Influenza B specific gene and 0.025 pM of reverse-tag primers, targeting an Influenza B specific gene. A concentration of 0.033 pM of MP specific to the tag used for Influenza B is also added to the reaction. Taq polymerase, Reverse Transcriptase, and components essential for their actions are added with Lyo-Ready 1-Step RT-qPCR Virus Master Mix (Meridian Bioscience™) according to the supplier's recommendations. An input volume of 5 pL of samples, synthetic DNA, quantified RNA or extracted from specific Influenza B strains, or water (negative control) are made in 15 pL of each reaction mix previously dispensed in a PCR plate.

[0219] The amplification program used on the Mic qPCR Cycler (Bio Molecular Systems) for the two reaction mixes tested is the same as previously described in Table 2.

[0220] The results observed in Figure 2 show the variability of fusion profiles between different samples of the same pathogen (Influenza B). These fusion temperature variations correspond to the genetic variability of the different strains of the pathogen. Therefore, it is complicated to justify that the fusion profiles of samples 723370 and 241027 correspond to specific signals of Influenza B detection which usually appear with a lower Tm on all synthetic DNA and RNA from viral strains. In contrast, the reaction mixes corresponding to the method of the invention shows that melting profiles all have the same melting temperature. In this way, the genetic variation within the same pathogen is attenuated by the method of the invention.

[0221] Moreover, the discrimination of different fusion profiles corresponding to different pathogens will be easier with the method of the invention because the groups will be much more distinct than with a classical melting method. Thanks to the method of the invention, it is possible to better discriminate the different target signals by limiting the impact of the evolution and genetic variations of the viral strains.

[0222] Example 3: Different Melting probes to differentiate the signals in a single reaction mix

[0223] In order to multiplex and discriminate several targets in a single reaction mix, it is essential to differentiate the detection signals with different Melting Probes. This example shows that it is possible to obtain specific signals by working with the labelled oligonucleotides (MP) described in Table 1, whose separation between quencher and reporter ranges from 15 to 55 nucleotides. In this example, the distance between the quencher and the reporter ranges from 15 to 55 nucleotides, but other distances lower than 15 and upper than 55 nucleotides may be tested.

[0224] The four reaction mixes using the method of the invention and targeting the synthetic DNA of a Meta pneumovirus are realized with the same way. Indeed, they contain 0.2 pM of tag-free forward primers targeting a specific gene of a Meta pneumovirus strain and 0.025 pM of reverse-tag primers, targeting a specific gene of Meta pneumovirus. Each MP is added to a reaction mix at the concentration of 0.033 pM. Taq polymerase and essential components are once again added with Lyo-Ready 1-Step RT-qPCR Virus Master Mix (Meridian Bioscience™) according to the supplier's instructions. An input volume of 5 pL of synthetic Metapneumovirus DNA or water are made into 15 pL of each reaction mix previously dispensed in a PCR plate.

[0225] The amplification program used on the Mic qPCR Cycler (Bio Molecular Systems) for the four reaction mixes tested is the same as described previously in Table 2.

[0226] The four reaction mixes show different melting profiles. Figure 3 shows four different melting profiles that can be distinguished from each other with their melting temperature. These distinct melting temperature signals are obtained by increasing the distance between the reporter and the quencher. Besides, the fluorescence signal of the reporter is correctly inhibited by the quencher if there is no specific target to detect when these two components are separated by 15 to 55 nucleotides. The method of the invention allows a fluorescence signal to be obtained correctly, no matter the distance between the reporter and the quencher is 15 or 55 nt.

[0227] 4: Use of different blockers on the 3' end of the Melting Probe (MP)

[0228] In order to avoid non-specific signals with the method of the invention, the 3' end of the melting probe (MP) must be blocked. This blocking can be done with different molecules that prevent the Taq polymerase from binding. The different oligonucleotides used in this example are described in Table 1.

[0229] These components are used to make six reaction mixes, each containing the specific MP of a synthetic Rhinovirus DNA with a different 3' blocker. All six mixes contain 0.1 pM of tag- free forward primers targeting a Rhinovirus strain-specific gene and 0.025 pM of reverse-tag primers, targeting a Rhinovirus strain-specific gene. Each Melting Probe is added to a reaction mix at the concentration of 0.033 pM. Taq polymerase and the components essential for its activity are again added with Lyo-Ready 1-Step RT-qPCR Virus Master Mix (Meridian Bioscience™) according to the supplier's recommendations. An input volume of 5 pL of synthetic Rhinovirus DNA or water are then put into 15 pL of each reaction mix previously dispensed into a PCR plate.

[0230] The amplification program used on the Mic qPCR Cycler (Bio Molecular Systems) for the six reaction mixes tested is the same as described previously in Table 2.

[0231] Figure 4 shows the fluorescence signals when the synthetic target DNA is added. This is not the case when water (negative control) is added instead. All of the MP used in this experiment are therefore quite specific for Rhinovirus, meaning that there is no loss of specificity with the change of the 3' blocker of the MP. There are also no significant differences between the amplification curves of the different reaction mixes tested, with the exception of the reaction mix containing the MP without a blocker on the 3' end. Indeed, the latter reaction mix allows a gain of about 4.5 Cq. However, not blocking the MP at its 3' end makes the signal more dependent on the target sequence. It is then difficult to control the fusion profile obtained between the targets, which makes it difficult to discriminate between them. Therefore, it is necessary to block the 3' end of the MP. However, although the sensitivity is not impacted by the change of blocker, it is possible to observe a difference in the fluorescence intensity as well as a shift in melting temperature between the different mixes. These observations simply highlight the fact that the different blockers may not have quite the same blocking characteristics. However, it confirms that the different types of blockers tested in 3' are functional.

[0232] It is also possible to use the quencher positioned at 5' directly as a blocker at 3'. This allows the two actions necessary for the functionality of the method of the invention to be mutualised. The experiment presented in figure 5 was carried out at the same time as the one presented above. The two reaction mixes are then made with the oligonucleotides presented in Table 1 , and used with the same components and in the same way as described for the previous example. The amplification program is also the same as that presented in Table 2.

[0233] Figure 5 shows that there is no significant difference in the fluorescence intensity of the fusion profiles between the two reaction mixes. Furthermore, the fusion profile obtained with the mix without modification on the 5' end is not modified and still allows the identification of a target without being affected by genetic variations. The amplification curves are also identical, which shows that the sensitivity is similar on the tested DNAs when the MP used has the quencher only in the 3' position. This alternative allows the sensitivity and specificity of the method of the invention to be maintained, while reducing the number of modifications to the melting probe. 5: Simultaneous detection of at least two nucleic acid

[0234] Probe

[0235] One of the advantages of the method of the invention is to allow the detection of several targets from the same reaction mix, and even simultaneously in some cases. Therefore, a multiplexing assay has been developed and is described in Figure 6. All the oligonucleotides used in this example are shown in Table 1.

[0236] To show that it is possible to perform the method in a multiplex, a single reaction mix is produced. This duplex targeting the synthetic DNA of a Metapneumovirus and a Rhinovirus. This reaction mix contains 0.2 pM of tag-free forward primers targeting a specific gene of a Metapneumovirus strain and 0.1 pM of tag-free forward primers targeting a specific gene of a Rhinovirus strain. It also contains 0.025 pM of each Reverse-tag primer, targeting specific Metapneumovirus and Rhinovirus genes. Each MP is added to the reaction mix at the concentration of 0.033 pM. Taq polymerase and essential components are added with Lyo- Ready 1-Step RT-qPCR Virus Master Mix (Meridian Bioscience™) according to the supplier's instructions. To finish, an input volume of 5 pL of Metapneumovirus synthetic DNA, Rhinovirus synthetic DNA, both synthetic DNA simultaneously or water (negative control) are made into 15 pL of each reaction mix previously dispensed into a PCR plate.

[0237] The amplification program used on the Mic qPCR Cycler (Bio Molecular Systems) for this example is the same as described in Table 2.

[0238] Figure 6 shows that it is possible to obtain signals from a reaction mix targeting simultaneously two infectious agents. Indeed, both melting profiles are observed as expected at temperatures of 79.7°C for Metapneumovirus and 82.8°C for Rhinovirus. It is also possible to obtain a characteristic melting profile when the two synthetic DNA are added simultaneously. Furthermore, there is no signal in the wells containing water (negative control), which shows that the specificity of the reaction is preserved, even with multiplexing. Therefore, these results demonstrated that it is possible to develop the method of the invention with the objective to add more targets and multiply them to detect more targets in the same reaction mix. The method of the invention is also capable of correctly discriminating the fusion profiles of two targets simultaneously. Then, it is possible to extend this method to a reaction mix containing more than two targets. Indeed, the method of the invention is particularly suitable for the detection of multiple targets, even within the same well.

[0239] Example 6: Use of different channels It is also possible to perform multiplex target detection using different channels. This example validates the possibility of the method of the invention to detect target by using a different fluorescent channel than the one used in the previous examples. The sequences of the Forward primer, Reverse-tag primer and MP used in this example are described in Table 1. Two reaction mixes were prepared. Each reaction is composed of 0.2 pM of tag-free target primers and 0.025 pM of target primers-tag. A concentration of 0.033 pM of Melting Probe (MP) specific to the target and the fluorescence channel used is also added to the reaction. Taq polymerase and the components essential for its action are added with Lyo-Ready 1- Step RT-qPCR Virus Master Mix (Meridian Bioscience™) according to the supplier's recommendations. An input volume of 5 pL of synthetic Meta pneumovirus DNA or water (negative control) are made in 15 pL of each reaction mix previously dispensed into a PCR plate.

[0240] The amplification of synthetic DNA by real-time PCR coupled with a melting step is performed on a Mic qPCR Cycler (Bio Molecular Systems) according to the amplification program shown above in Table 2.

[0241] Figure 7 shows that it is possible to obtain amplification curves and melting profiles on the HEX channel for the detection of Metapneumovirus. The melting profile obtained with the MP containing the HEX fluorophore on the HEX channel is very similar to the one obtained with the MP containing the FAM fluorophore on the FAM channel. Indeed, the melting temperatures are respectively 79.6°C and 79.9°C for the MP containing HEX and FAM. Whatever the fluorophore used, the Cq obtained for the detection of Metapneumovirus is 34. Therefore, there is no sensitivity loss between using the method with a HEX or FAM fluorochrome. This means that the method of the invention can be used with different other fluorophores. As a results, different targets can be detected using different fluorescence channels.

[0242] It is then possible to combine the detection of several targets on the same channel (example 5) with the detection of several targets on different channels (example 6). In this way, the method the invention allows the detection of a multitude of targets from several fluorescence channels.

[0243] 7: Combination with real-time PCR detection a Taq Man

[0244] The detection technology of the invention can be used in combination with real-time PCR detection using a TaqMan probe. The oligonucleotides' sequences (Forward, Reverse and Reverse-tag primers) and probes (Melting Probe and TaqMan probe) used in this example are described in Table 1. A single reaction mix was performed. Each reaction consists of 0.1 pM of tag-free Forward primer targeting Rhinovirus DNA and 0.025 pM of tag Reverse primer targeting Rhinovirus DNA, and 0.2 pM of tag-free Forward primer targeting Meta pneumovirus DNA, and 0.1 pM of tag- free Reverse primer targeting Metapneumovirus DNA. A concentration of 0.033 pM Melting Probe and TaqMan probe targeting Rhinovirus and Metapneumovirus DNA respectively are added to the reaction. Taq polymerase, Reverse Transcriptase and the components essential for their actions are added with Lyo-Ready 1-Step RT-qPCR Virus Master Mix (Meridian Bioscience™) according to the supplier's recommendations. An input volume of 5 pL of synthetic Rhinovirus, Metapneumovirus or water (negative control) DNA are then made in 15 pL of each reaction mix previously dispensed into a PCR plate.

[0245] The amplification of synthetic DNA by real-time PCR coupled with a melting step, itself coupled with TaqMan detection, is performed on a Mic qPCR Cycler (Bio Molecular Systems) according to the amplification program shown in Table 2 above.

[0246] The results observed in Figure 8 show that it was possible to obtain differential detection between Rhinovirus and Metapneumovirus DNA using the technology of the invention and TaqMan technologies within the same well. Indeed, a melting profile characteristic of Rhinovirus DNA detection is visible from the FAM channel with a melting peak at 83°C. Furthermore, the amplification curves corresponding to this detection of Rhinovirus DNA are visible on this same FAM channel. However, it is not possible to observe a FAM melting profile when Metapneumovirus DNA is added to the well. A fluorescence signal corresponding to hydrolysis of the TaqMan probe targeting Metapneumovirus DNA is detected on the CAL Fluor red 610 channel. This signal corresponds to a conventional realtime PCR amplification signal.

[0247] Therefore, different targets can be detected in the same well using these two different technologies. This allows to offer increased multiplexing, as well as the possibility to specifically quantify a target if required.

[0248] Example 8: Gain of sensitivity of the MPM technology of the invention in comparison to classical hybridization

[0249] The aim of this example was to compare the signal obtained from the formation of a duplex following an elongation step (‘partial hybridization with elongation’) with the signal obtained from a classical hybridization of the probe to the neosynthesized strand (‘total hybridization without elongation’).

[0250] The sequences of the oligonucleotides (Forward, and Reverse-tag primers) and probes (MP and TaqMan probe) used in this example and targeting a Coronavirus B specific gene are described in Table 1 above. They said oligonucleotides were designed based on Human Coronavirus HCoVs OC43 and HKU1.

[0251] Two reaction mixes were produced. For both mixes, each reaction is made with 0.1 pM of each of the ‘Forward primers without tag’ targeting Coronavirus B specific gene, 0.025 pM of ‘Reverse primers with tag’ targeting Coronavirus B specific gene, and 0.025 pM of Melting Probe allowing Coronavirus B detection. Taq polymerase, Reverse Transcriptase and the components essential to their actions are added with Lyo-Ready 1-Step RT-qPCR Virus Master Mix (Meridian Bioscience™) according to the supplier's recommendations. An input volume of 5 pL of synthetic Coronavirus B DNA are then made in 15 pL of each reaction mix previously distributed in a PCR plate.

[0252] Amplification of the synthetic DNA by real-time PCR coupled with a melting step is performed on a Mic qPCR Cycler (Bio Molecular Systems) according to the amplification program shown in Table 2 above.

[0253] The figure 9 illustrates the fluorescence signals and amplification curves obtained with the two methods (MPM technology of the invention and classical hybridization). A melting profile at 78.6°C and an amplification curve were obtained when the complementary neosynthesized strand of the tag hybridizes partially (tag-sept) or totally (tag-neuf) to the fluorescent probe. In the first case (invention), the forming duplex producing a fluorescent signal requires Taq polymerase-mediated elongation of the tag's neosynthesized strand (partial hybridization with elongation), whereas in the second case (classical hybridization) the duplex is formed immediately (total hybridization without elongation). The results showed that the obtained Cq (quantification cycle) are respectively 31.5 for the partial hybridization probe (with elongation) and 33 for total hybridization probe (without elongation). These results demonstrated that the MPM technology with elongation caused using a neosynthesized partial hybridization tag on the fluorescent probe provides a non-negligible gain in sensitivity of around 1.5 Cq in the present example, in comparison to classical hybridization (total hybridization). And this gain of sensitivity has a positive impact on the detection of a target gene in a sample. So all these results demonstrated that the method of the invention, wherein the forming duplex producing a fluorescent signal requires Taq polymerase-mediated elongation of the tag's neosynthesized strand, provides more flexibility on the design of the primers-tags and probes, in terms of length, concentration in GC, Tm and is particularly adapted to multiplex embodiments.

[0254] REFERENCES

[0255] Gatcombe et al. 2010. Evaluation of a molecular beacon real-time PCR assay for detection of Baylisascaris procyonis in different soil types and water samples. Parasitol Res. (2010) 106:499-504.

[0256] Liu et al., 2019, Detection of 12 Common Food-Borne Bacterial Pathogens by TaqMan Real-Time PCR Using a Single Set of Reaction Conditions. Frontiers in Microbiology. February 2019, Volume 10, Article 222.

[0257] Pereira et al., 2014, Neospora caninum DNA detection by TaqMan real-time PCR assay in experimentally infected pregnant heifers. Vet Parasitol., 2014 Jan 31 ; 199(3- 4):129-35. doi: 10.1016 / j.vetpar.2013.10.018. Epub 2013 Oct 30.

[0258] Phung et al, 2013. Rapid Detection and Identification of Nontuberculous Mycobacterial Pathogens in Fish by Using High-Resolution Melting Analysis. Applied and Environmental Microbiology. December 2013. Volume 79, Issue 79, Number 24, p. 7837-7845.

[0259] Reed 2007. High-Resolution DNA melting analysis for simple and efficient molecular diagnostics. Pharmacogenomics (2007), 8(6), 597-608.

[0260] Salvatore et al. 2021. A Molecular-Beacon-Based Multiplex Real-Time PCR Assay To Distinguish Mycobacterium abscessus Subspecies and Determine Macrolide Susceptibility. Journal of Clinical Microbiology. Volume 59, Issue 8, e00455-21.

Claims

CLAIMS1. An in vitro method of detecting at least two target nucleic acid sequences from a biological sample comprising: a) releasing and / or extracting the nucleic acids from the biological sample, and b) amplifying and detecting at least two target nucleic acid sequences in a PCR reaction using the nucleic acids of step a) as a template, wherein the DNA amplification and detection step b) comprises:(b1) hybridizing each target nucleic acid sequence with a specific primers pair composed of a forward primer and a reverse primer comprising nucleotide sequences complementary to the target nucleic acid sequence, wherein at least one primer of the pair additionally comprises a tag sequence that is linked to its 5’-end portion;(b2) contacting the resultant of step b1) with a template-dependent nucleic acid polymerase under primer extension conditions for inducing the 3’-extension reaction and forming an extended neosynthetized strand;(b3) denaturing the amplified product of step (b2) and contacting the extended neosynthetized single-strand with a probe sequence comprising a 3’ blocker and an interactive label, wherein the probe sequence hybridizes with the 3’-end portion of the extended neosynthetized single-strand and forms, under conditions for inducing the 3’-extension reaction, an extended duplex providing the signal; and c) optionally quantifying the amount of the said target nucleic acids.

2. The in vitro method of claim 1 , wherein the tag sequence has a linear structure with no interactive label, and wherein the probe sequence comprises a fluorophore and a quencher.

3. The in vitro method of claim 1 or 2, wherein the distance between the fluorophore and the quencher on the probe sequence ranges from 5 to 70 nucleotides, in particular 10 to 60 nucleotides, preferably 15 to 55 nucleotides.

4. The in vitro method of claim 1 , wherein the 3’ blocker is the quencher.

5. The in vitro method according to anyone of claims 1 to 4, wherein the length of the tag sequence linked to the 5’end portion of at least one primer, ranges from 8 to 30 nucleotides, preferably from 10 to 20 nucleotides, and wherein the length of the probe sequence ranges from 20 to 100 nucleotides, in particular from 30 to 60 nucleotides.

6. The in vitro method according to anyone of claims 1 to 5, wherein the primers-tag and / or the probes have modifications in their conformation and / or their composition reducing thecompetition between primer-tag, probe and complementary strand of the neosynthesized strand and / or increasing the melting temperature of the 3’ end portion of the probe sequence to promote the binding of the complementary neosynthesized strand with the 3’ end portion of the probe, in particular selected in the group consisting of a primer-tag and / or probe having a hairpin structure, a 5’-Primer-tag modification including 5’-Phosphate, 5’-Amino groups, or 5’-Biotin, and / or a a primer-tag and / or probe having LNA modification.

7. The in vitro method according to anyone of claims 1 to 6, wherein the biological sample is selected in the group consisting of a sample from an animal or human subject, in particular a sample of bodily fluid such as blood and its derivatives, urine, saliva, faeces, milk, mucous secretions, nasopharyngeal or oropharyngeal swabs, bronchoalveolar fluids, trans-tracheal aspirates, a sample of tissue or an organ, or an eluate from rag, a sample derived from microbial culture, seeds or other plant tissues, or a sample of environment.

8. The in vitro method according to anyone of claims 1 to 7, wherein the target nucleic acids are selected from the group consisting of nucleic acids from the genome of different animal species, human, microorganisms, or plant species, in particular (i) nucleic acids sequences correlated to genetic or phenotypic treats of interest, (ii) mutations or variations of nucleic acids sequences correlated to a physiological or pathological disorder, (iii) nucleic acids sequences of a pathogen that may have infected a subject or a sample of environment, and (iv) mixtures thereof, such as genomic nucleic acids of the subject and / or nucleic acids of pathogens having infected the subject, or nucleic acids of pathogens present in a sample of environment in particular nucleic acids of virus, bacteria, fungi, yeast, parasite, and mixtures thereof .

9. The in vitro method according to claim 8, wherein the target nucleic acids are DNA or RNA of a virus, in particular selected from respiratory virus such as influenza virus, respiratory syncytial virus, parainfluenza viruses, metapneumovirus, rhinovirus, coronaviruses, adenoviruses, and bocaviruses, or a Human Immunodeficience Virus (HIV), or DNA of bacteria strain such as bacterial strains causing bovine mastitis.

10. The in vitro method according to anyone of claims 1 to 9, wherein the PCR reaction is a qPCR coupled with melting analysis.

11. The in vitro method according to anyone of claims 1 to 10, wherein the PCR reaction is a multiplex PCR reaction comprising at least two primer pairs targeting at least two target nucleic acids and a tag sequence per primers pair, for amplifying, detecting and optionally quantifying, simultaneously, at least two target nucleic acids, preferably a multiplex PCR reaction coupled with melting analysis.

12. The in vitro method according to anyone of claims 1 to 11 , wherein the multiplex PCR reaction is carried out with 2 to 25 target nucleic acids, in particular 2 to 10 target nucleic acids, either in a sole channel or in several channels.

13. The in vitro method according to anyone of claims 10 to 12 wherein the melting analysis is automatized and coupled with a method for classification of target signals of a biological sample comprising: acquisition of at least one DNA melting measurement curve of the biological sample, determination that the measurement biological sample belongs to a defined group among different possible groups, by analysis of descriptors originating from the at least one measurement curve, characterized in that the descriptors comprise one or more points of the first and / or second derivative of each measurement curve.

14. Use of a kit in the in vitro method of any one of claims 1-13 for detecting at least two target nucleic acid sequences from a biological sample, which comprises : a) a specific primers pair for each target nucleic acid sequence, wherein at least one primer of the pair additionally comprises a tag sequence that is linked to its 5’-end portion; b) a template-dependent nucleic acid polymerase capable of inducing the 3’-extension reaction at the 3’-end of the primer and forming an extended neosynthetized strand; c) at least one probe sequence comprising a 3’ blocker and an interactive label, wherein the probe sequence hybridizes with the 3’-en portion of the extended neosynthetized singlestrand ; d) optional reagents for denaturing the amplified products and performing target amplification PCR such as buffers, DNA polymerase cofactors, deoxyribonucleotide-5-triphosphates, and reverse transcriptase ; e) optional reagents necessary for performing positive and negative control reactions ; and f) optional reagents for detecting the extended duplex formed with the probe sequence comprising the interactive label.

15. Use of the in vitro method according to anyone of claims 1 to 13 or use of the kit according to claim 14 for (i) detecting and optionally quantifying nucleic acids target sequence(s) in a biological sample such as animal, human, plant, or environmental sample, and / or (ii) screening and selecting genes or markers of interest, notably in the domains of health, virology, agriculture, ecological or environmental field, in particular detecting and optionally quantifying, nucleotide variations or mutations in a subject, such as variationscorrelated with genetic or phenotypic traits of the subject , or variations or mutations correlated with physiological disorders of a subject, and / or detecting and optionally quantifying nucleic acids of pathogens having infected the subject or pathogens present in a sample of environment.

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