Isothermal amplification in animal samples

Abstract

The present invention relates to isothermal amplification techniques and methods for the detection of nucleic acids in animal samples.

Description

ISOTHERMAL AMPLIFICATION IN ANIMAL SAMPLESFIELD OF INVENTION

[0001] The present invention relates to isothermal amplification techniques and methods for the detection of nucleic acids in animal samples, in particular non-human animal samples.BACKGROUND OF INVENTION

[0002] Isothermal amplification techniques, such as Loop-mediated isothermal AMPlification (LAMP), are of great use for the rapid detection of infectious agents in veterinary diagnostics, as they have comparable sensitivity and specificity to PCR, and require little material and time to be carried out.

[0003] The LAMP technique is a DNA amplification technique that uses a strand displacement polymerase. Advantageously, the amplification may thus proceed at room temperature, as it is no longer necessary to dehybridize the DNA at a higher temperature, because the dehybridization step is carried out by an enzymatic process.

[0004] According to one variant of the LAMP method, six primers recognize eight distinct regions of the target DNA. Observation of the amplicon produced can be carried out by fluorescence by adding to the reaction a DNA intercalator which emits fluorescence in the presence of double-stranded DNA.

[0005] The LAMP technology, and other isothermal amplification techniques, may thus be adapted for point-of-care tests, when coupled to rapid nucleic acid extraction and purification.

[0006] There remains a need for methods of detection and / or quantification of nucleic acids in animal samples, especially non-human animal samples, for example cats / felids and horses / equids.

[0007] In particular, there remains a need for methods which are applicable to, both, domestic animals and non-domestic animals; for example large animals (e.g. livestock).

[0008] In particular, there remains a need for methods which are applicable for point-of- care tests, in a reliable and cost-effective manner.

[0009] In particular, there remains a need for methods which are capable of discriminative true-positive samples from false-positive samples.

[0010] The invention has for purpose to meet the above-mentioned needs.SUMMARY

[0011] According to a first main embodiment, the invention relates to an in vitro method for detecting nucleic acids in a non-human animal sample, comprising steps of: a) providing a nasal, oral or oropharyngeal non-human animal sample susceptible to contain a nucleic acid of interest, or a fraction thereof; b) detecting a modulation of fluorescence intensity on or after isothermal amplification of the sample, thereby detecting an amplified fraction; c) detecting a modulation of fluorescence intensity as a function of temperature in the amplified fraction, and comparing it to a reference value, thereby detecting the nucleic acid of interest.

[0012] According to a second main embodiment, the invention relates to an in vitro method for discriminating true-positive isothermal amplification from false-positive isothermal amplification in a non-human animal sample, comprising steps of:a) providing a nasal, oral or oropharyngeal non-human animal sample susceptible to contain a nucleic acid of interest, or a fraction thereof; b) detecting a modulation of fluorescence intensity on or after isothermal amplification of the sample or fraction thereof, thereby detecting an amplified fraction; c) detecting a modulation of fluorescence intensity as a function of temperature in the amplified fraction, and comparing it to a reference value, thereby discriminating true-positive isothermal amplification from false-positive isothermal amplification.

[0013] According to a third main embodiment, the invention relates to an in vitro use of melting curve analysis for discriminating true-positive isothermal amplification from false-positive isothermal amplification in a nasal, oral or oropharyngeal non-human animal sample, or a fraction thereof.DEFINITIONS

[0014] In the present invention, the following terms have the following meanings:

[0015] “About” preceding a figure means plus or less 10% of the value of said figure.

[0016] “And / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0017] “At least one” includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 250, 500, 750, 103,104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015or more.

[0018] “Comprising”, “comprises” and “comprised of’ are used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. These terms also encompass “consisting of’.

[0019] “Nucleic acid” or “polynucleotide” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Nucleic acid” or “Polynucleotides” include, without limitation single-and doublestranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double- stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, “Nucleic acid” or “polynucleotide” refers to triplestranded regions comprising RNA or DNA or both RNA and DNA. The term “nucleic acid” or “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, “nucleic acid” or “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short polynucleotides, often referred to as oligonucleotides. Nucleic acids may, for example, consist of deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), including messenger ribonucleic acids (mRNAs), transfer ribonucleic acids (tRNAs), ribosomal ribonucleic acids (rRNAs).

[0020] “Protein”, “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labelling component. As used herein the term “amino acid” includes natural and / non-natural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

[0021] “Nucleic acid extraction” refers to the action of physically separating nucleic acids from other constituents of the biological entity that contains them. Since nucleicacids are contained in biological entities and protected from the environment by membranes and / or walls systems, in particular cell membranes and / or walls, the extraction of nucleic acids generally comprises a step of breaking or solubilizing the cell membranes and / or walls and releasing the nucleic acids into the reaction mixture; these phenomena are also defined as lysis.

[0022] “Sample” refers to a biological material obtained from an animal body, from an environment, or from a food product, preferably, obtained beforehand by an appropriate sampling technique on an individual or a product. By extension, “Complex sample” relates to a sample which can comprise, in addition to the nucleic acids of interest, other constituents, such as polypeptides, lipids, polysaccharides, salts, metals, trace elements, solvents, acids, bases, and the likes. A sample may, for example, be selected from a non- exhaustive list comprising, or consisting of, bile, feces, aqueous humor, milk, amniotic fluid, lymph, cerebrospinal fluid, plasma, product of respiratory lavage, product of a throat pouch, pus, nasopharyngeal secretion, lacrimal secretion, vaginal secretion, saliva, blood, serum, semen, and urine.

[0023] As used herein, the term “nasopharyngeal sample” refers both to “nasal samples” and “oropharyngeal samples”. Hence, the term may refer to any biological material obtained from, or derived from, the upper respiratory tract, such as product of respiratory lavage. Examples of nasopharyngeal samples, according to the present disclosure, may thus comprise or consist of a sample selected from the group consisting of: nasopharyngeal swab, nasopharyngeal wash, sinus secretions, bronchoalveolar lavage, endotracheal aspirate, and combinations thereof.

[0024] As used herein, a “product of respiratory lavage” includes a tracheal lavage, a bronchoalveolar lavage and a pulmonary lavage. In some embodiments, lavage and wash are intended to be equivalent terms.

[0025] As used herein, the term “oral sample” may refer to any biological material obtained from, or derived from, the oral cavity, for example any material selected from the group consisting of: oral blood, oral plaque, oral plasma, saliva, sputum, buccal cavity fluid, buccal cells, and compositions thereof.

[0026] “Amplification” refers to the specific multiplication of the number of copies of the nucleic acids of interest.

[0027] As used herein, the term “isothermal nucleic acid amplification” or “isothermal amplification” refers to nucleic amplification methods which may the achieved isothermally such as, for example, without a change in temperature of a reaction mixture. Hence, they may comprise or consist of non-thermal cycling dependent amplification methods. Isothermal amplification generally requires a set of probes. For example, For example, Loop-Mediated Amplification generally requires at least four oligonucleotide probes (or “primers”), but under certain variants may further comprise additional probes to improve amplification efficiency, such as “loop primers”, which may result in a total of at least six primers per target sequence. A method for isothermal nucleic acid amplification described herein may be completed without cycling the temperature of an amplification reaction mixture. For example, multiple amplification cycles may be performed without cycling the temperature of a reaction mixture. Isothermal amplification methods may include, in a non-exhaustive manner, those selected from the group consisting of: Nicking and Extension Amplification Reaction (NEAR), Rolling Circle Amplification (RCA), Helicase-Dependent Amplification (HDA), Loop-Mediated Amplification (LAMP), Transcription-Mediated Amplification (TMA), Self-Sustained Sequence Replication (3SR), Nucleic Acid Sequence Based Amplification (NASBA), Single Primer Isothermal Amplification (SPIA), Q-|3 Replicase System, Strand Displacement Amplification (SDA), Multiple Displacement Amplification (MDA), and Recombinase Polymerase Amplification (RPA). Typically, isothermal methods require a set temperature, which is determined by the reagents being used. For example, in LAMP the enzymes function best between 60 and 65° C. The amplification reaction can generally be followed in real-time, directly or indirectly, either by measuring the turbidity or by fluorescence using, for example, intercalating dyes (such as ethidium bromide, SYBR Green, Eva Green and YO-PRO-I) and / or fluorescent labeled probes (or “primers”). Dye molecules intercalate or directly label the nucleic acids (e.g. DNA), and in turn can be correlated to the number of copies initially present. Generally, the intercalating dye is a non-sequence-specific fluorescent dye that exhibits a large increase in fluorescence emission upon binding to nucleic acid, such as double-strandedDNA (dsDNA). Using these properties, nucleic acid amplification can be monitored in real time by continuously measuring fluorescence during the LAMP reaction. Fluorescence-based detection of isothermal amplification (e.g. LAMP) may also depend on the mechanism of Forster resonance energy transfer (FRET) (Chen Q et al., 1997 Biochemistry 36 (15): 4701-11).

[0028] As used herein, the terms “melting curve”, "thermal melting curve", "thermal melt curves", “annealing curve”, "thermal property curve", "thermal denaturation curve" or "thermal profile curve”, are considered as synonymous. They refer to a denaturation of a double stranded nucleic acid (for example a partially or completely double-stranded nucleic acid resulting from an amplification reaction, such as dsDNA) in the presence of a fluorescent compound (e.g. an intercalating dye or a labeled nucleic acid probe) which is indicative of binding of the two strands corresponding to said nucleic acid. The partially or completely double-stranded nucleic acid may thus comprise our consist of an amplified nucleic acid, either single-stranded or double-stranded, in complex with the fluorescent compound. The term is not meant to be limitative toward one specific fluorescent compound. Thus, by determining the fluorescence intensity as a function of temperature, information is gained on the double-stranded nucleic acid; in particular with respect to its corresponding melting temperature (Tm). In a non-exhaustive manner, the modulation of fluorescence intensity as a function of temperature, may for example be expressed as:A fluorescence intensity as a function of temperature;A derivative of fluorescence intensity as a function of temperature (or “ derivative melt plot

[0029] “Individual” refers to an animal, vertebrate or invertebrate, preferably a mammal animal. The mammal animal may be a non-human mammal or a human.

[0030] As used herein, the term “Felidae” includes all members of the family of mammals in the order Carnivora colloquially referred to as “cats”. In a non-exhaustive manner, the term may thus encompass non-human mammals, whether domestic or not, selected from the group consisting of: domestic cats and non-domestic cats, such as lions, cheetahs, panthers, cougars, and leopards. Hence, the term may encompass the “domesticcat”, or “house cat”, or Felis catus and other non-domestic species in the family Felidae, unless stated otherwise.

[0031] As used herein, the terms “equine” and “equine animal” may be used interchangeably, and include any member of the genus Equus; which may thus include horses, asses and zebras. It encompasses, e.g., any horse or pony, the taxonomic designations Equus ferus and / or Equus caballus. and / or the subspecies Equus ferus caballus. The equine animal may e.g., be a domestic horse.

[0032] As used herein, the term “neutral pH” includes a pH ranging from about 6.5 to about 9.0, preferably from about 7.0 to about 8.5, preferably from about 7.5 to about 8.0.

[0033] As used herein, the term “acidic pH” refers to a pH below 6.5. In some alternative embodiments, step a) and / or optionally step b) is / are performed under basic pH. As used herein, the term “basic pH” refers to a pH above 9.0.

[0034] As used herein, and without otherwise statements, concentrations expressed as a percentage (%) refer to concentration as weight / volume (w / v), in particular as g / 100 mL. Illustratively, an SDS concentration ranging from about 1% to about 25% is understood to refer to an SDS concentration ranging from about 1 g / 100 mL to about and 25 g / 100 mL.DETAILED DESCRIPTION

[0035] The inventors sought to determine whether using melting curves after LAMP amplification phase could distinguish nonspecific from specific amplifications for feline respiratory pathogens (feline herpesvirus, feline calicivirus, and Chlamydia felts') causing Coryza syndrome (eye discharge, nasal discharge, inflammation of the respiratory tract, sneezing, etc.), and equine pathogens (Equine Herpesvirus-1 (EHV-1), Equine Herpesvirus-4 (EHV-4), Influenza, and Streptococcus equi).

[0036] Surprisingly, the inventors have now identified that certain nucleic acid samples, such as oropharyngeal swabs, generate nonspecific isothermal amplification signal, when the amplification signal is detected by measuring a modulation of fluorescence.

[0037] Unfortunately, non-specific amplification can be responsible for the appearance of a weak or strong amplicon signal, even in the absence of the targeted nucleic acid in the sample. This may lead to an increase of false-positive LAMP results; thereby reducing the sensitivity of the test.

[0038] Without wishing to be bound by the theory, the inventors are of the opinion that the increase of false-positives is due to unforeseen auto-fluorescence properties in animal samples, in particular non-human animal samples; and more particularly nasal, oral or oropharyngeal samples, such as oropharyngeal swabs, or fractions thereof.

[0039] Auto-fluorescence properties of some plant samples have been reported in the Art, in the context of isothermal amplification. Dickinson (“Loop-Mediated Isothermal Amplification (LAMP) for Detection of Phytoplasmas in the Field”, Methods Mol Biol. 2015) reports a nucleic acid-based detection method applied to samples from Napier grass (Pennisetum purpureum). In this study, it was found an apparent increase in fluorescence in the first few minutes of the amplification, with an atypical shape.

[0040] However, to the knowledge of the inventors, this auto-fluorescence has not yet been reported in other non-plant samples; let alone in non-human animal samples such as cat or equine samples.

[0041] Unfortunately, such unforeseen auto-fluorescence properties are susceptible to reduce the sensitivity and reproducibility of tests relying on a detection of a fluorescent signal, to the extent that false-positives may occur.

[0042] Hence, this could ultimately be also highly detrimental to their use for detecting an underlying disorder and / or infection, in a reliable manner, especially in a veterinary context (e.g. for the follow-up of farm or domestic animals).

[0043] The artefactual fluorescence signal would also be particularly detrimental to isothermal nucleic acid amplification methods, which heavily rely on the measurement of a fluorescence signal for establishing an amplification curve.

[0044] Consequently, the inventors describe hereafter isothermal amplification methods for detecting nucleic acids in nasal, oral or oropharyngeal samples, and for discriminating true- and false-positive isothermal amplifications in such samples, with increased sensitivity and reproducibility.

[0045] More particularly, the inventors propose to rely on post-amplification melting curve as a quality control, for evaluating an isothermally amplified fraction in non-human animal samples. Post-amplification melting curves generally rely on a modulation of a signal (e.g. a fluorescence signal), for example a derivative of said signal, as a function of temperature; whereas isothermal amplification refers to experimental conditions wherein the temperature of the amplification reaction is typically constant or nearly constant, unlike polymerase chain reaction (PCR).

[0046] According to a first main embodiment, the invention relates to an in vitro method for detecting nucleic acids in a non-human animal sample, comprising steps of a) providing a nasal, oral or oropharyngeal non-human animal sample susceptible to contain a nucleic acid of interest, or a fraction thereof; b) detecting a modulation of fluorescence intensity on or after isothermal amplification of the sample or fraction thereof, thereby detecting an amplified fraction; c) detecting a modulation of fluorescence intensity as a function of temperature in the amplified fraction, and comparing it to a reference value, thereby detecting the nucleic acid of interest.

[0047] According to a second main embodiment, the invention relates to an in vitro method for discriminating true-positive isothermal amplification from false-positive isothermal amplification in a non-human animal sample, comprising steps ofa) providing a nasal, oral or oropharyngeal non-human animal sample susceptible to contain a nucleic acid of interest; b) detecting a modulation of fluorescence intensity on or after isothermal amplification of the sample, thereby detecting an amplified fraction; c) detecting a modulation of fluorescence intensity as a function of temperature in the amplified fraction, and comparing it to a reference value, thereby discriminating true-positive isothermal amplification from false-positive isothermal amplification.

[0048] Advantageously, a modulation of fluorescence intensity, as a function of temperature, in the amplified fraction, can be indicative of a false-positive when the corresponding melting temperature derivable from the modulation in the sample is different from the melting temperature derivable from the modulation in the reference.

[0049] Depending on the experimental protocol, the nature of the amplified fraction and the sensitivity of the test, the man skilled in the Art may thus consider which type of reference is optimal for a given isothermal amplification.

[0050] The proposed methods are thus particularly amenable to farm and domestic animals, for which nasal, oral or oropharyngeal samples can be collected.

[0051] According to particular embodiments, the nasal, oral or oropharyngeal samples can be collected by rubbing, wiping or swabbing according to good practice and protocols acknowledged in the state of the art.

[0052] In certain embodiments, the samples are collected in compliance with good practices and possibly with the regulatory standards in force, in particular in veterinary medicine. For example, the conditions of sterility may be desired. Samples may be collected using a suitable instrument, including but not limited to, syringe, toothpick, swab, spatula, receptacle, forceps, scalpel, adhesive paper.

[0053] In practice, the non-human sample, according to the present disclosure, can be of very varied origin. According to particular embodiments, the sample is from a non-humananimal; preferably a mammal. In certain embodiments, the mammal is selected from the non-exhaustive list comprising, or consisting of, a cat, a horse, a goat, a dog, a guinea pig, a rabbit, a sheep, a pig, a rat, a mouse, a cow, a chicken. In some embodiments, the mammal is a pet selected from the non-exhaustive list comprising, or consisting of, a cat, a dog, a guinea pig, a rat, a mouse. In certain embodiments, the mammal is an animal of economic interest, for example selected from the non-exhaustive list comprising, or consisting of, a horse, a rabbit, a sheep, a pig, a cow, a poultry.

[0054] According to exemplified embodiments, the non-human animal sample is a cat sample. According to other exemplified embodiments, the non-human animal sample is an equine sample.

[0055] According to particular embodiments, the non-human animal sample is an oral sample; more particularly a swab sample. According to a particular embodiment, the non- human animal sample is an oral cat sample or an oral equine sample; for example a cat swab sample or an equine swab sample.

[0056] According to another particular embodiment, the non-human animal sample is a nasopharyngeal cat sample or a nasopharyngeal equine sample.

[0057] According to some non-mutually exclusive embodiments, the non-human animal sample may further contain plant cells. According to some non-mutually exclusive embodiments, the non-human animal sample is devoid of plant cells.

[0058] According to some non-mutually exclusive embodiments, the non-human animal sample may contain only one cell type. According to some non-mutually exclusive embodiments, the non-human animal sample may contain a plurality of cell types. In a non-exhaustive list, the non-human animal sample may contain one or more of the following cell types: an adipocyte; a fibroblast; an endothelial cell; an epithelial cell; a bone cell, e.g., an osteoblast, an osteocyte, an osteoclast; a muscle cell, e.g., a myoblast, a myocyte; a blood cell, e.g., a red blood cell, a lymphocyte, a polynuclear; a hepatocyte; a keratinocyte; a nerve cell; and any combination thereof.

[0059] According to particular embodiments, the modulation (e.g. decrease or increase) of fluorescence intensity is detected by measuring a modulation of a fluorescence signal from one or more intercalating dyes and / or fluorescent probes, such as labeled nucleic acid probes.

[0060] According to particular embodiments, the modulation of fluorescence intensity as a function of temperature is a fluorescence intensity as a function of temperature. According to other particular embodiments, the modulation of fluorescence intensity as a function of temperature is a derivative of fluorescence intensity as a function of temperature.

[0061] According to particular embodiments, the modulation of fluorescence intensity as a function of temperature in the amplified fraction, is achieved by modulating (e.g. decreasing) the temperature in the amplified reaction, from a temperature higher or substantially higher than the temperature at which isothermal amplification did occur, to a temperature lower or substantially lower than the temperature at which isothermal amplification did occur.

[0062] For example, the modulation of fluorescence intensity as a function of temperature in the amplified fraction, may be achieved by decreasing the temperature of the amplified fraction from about 90°C or more, to a temperature lower or substantially lower than the temperature at which isothermal amplification did occur.

[0063] According to particular embodiments, the modulation of fluorescence intensity as a function of temperature in the amplified fraction, is achieved by modulating the temperature of the amplified fraction in a range from about 50°C to about 98°C, for example from about 60°C to about 98°C.

[0064] According to particular embodiments, the modulation of fluorescence intensity as a function of temperature in the amplified fraction, is achieved by modulating the temperature of the amplified fraction in a range from about 95°C to about 65°C or from about 98°C to about 60°C or from about 92°C to about 65°C. For example, the modulation of fluorescence intensity as a function of temperature in the amplified fraction, is achieved by modulating the temperature of the amplified fraction in a range from about 98°C toabout 60°C; for example from about 98°C, 97°C, 96°C, 95°C, 94°C, 93°C, 92°C, 91°C or 90°C to about 85°C, 84°C, 83°C, 82°C, 81°C, 80°C, 79°C, 78°C, 77°C, 76°C, 75°C, 74°C, 73°C, 72°C, 71°C, 70°C, 69°C, 68°C, 67°C, 66°C, 65°C, 64°C, 63°C, 62°C, 61°C, ot 60°C.

[0065] Advantageously, for fluorescence measurement, a read can be obtained for 10 seconds or less.

[0066] According to some embodiments, melting curve analysis is achieved in the form of High-Resolution Melt (HRM) analysis. HRM analysis generally requires data collection in narrow temperature increments; for example, for example a step of heating the amplification product at a rate of l°C / s or less, for example a step of heating the amplification product at a rate of 0.002°C / s to l°C / s or more. According to some embodiments, melting curve analysis is not achieved in the form of High-Resolution Melt (HRM) analysis; for example the melting curve analysis is achieved by a step of heating the amplification product at a rate of more than, or substantially more than, l°C / s.

[0067] According to particular embodiments, the amplified nucleic acid of interest may be selected from the group consisting of deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), including messenger ribonucleic acids (mRNAs), transfer ribonucleic acids (tRNAs), ribosomal ribonucleic acids (rRNAs).

[0068] According to particular embodiments, the nucleic acid(s) of interest(s) is / are exclusively deoxyribonucleic acids (DNAs). According to particular embodiments, the nucleic acids are exclusively ribonucleic acids (RNAs). According to particular embodiments, the nucleic acids are a mixture of deoxyribonucleic acids (DNAs) and ribonucleic acids (RNAs).

[0069] According to particular embodiments, the nucleic acid(s) of interest(s) is / are double-stranded deoxyribonucleic acids (DNAs).

[0070] In certain embodiments, the nucleic acids are selected in the group consisting of a genomic nucleic acid and an extragenomic nucleic acid. Non-limitative examples of genomic nucleic acids include viral genomes and chromosomes, such as bacterialchromosomes, bacterial artificial chromosomes (BAC), eukaryotic chromosomes, and the likes. Non-limitative examples of extragenomic nucleic acids include plasmids, cosmids, viral vectors, and the likes. In some embodiments, the nucleic acids are genomic nucleic acids.

[0071] According to particular embodiments, the reference value corresponds to, or is indicative of, a melting temperature corresponding to one or more reference nucleic acids, in particular reference double-stranded nucleic acids, more particularly double-stranded deoxyribonucleic acids (dsDNA), in the amplified fraction.

[0072] According to particular embodiments, the reference value is determined on a reference sample comprising the nucleic acid of interest.

[0073] According to particular embodiments, the isothermal amplification may be carried out at a constant temperature of about 50°C, 55°C, 60°C, 60.5°C, 61°C, 61.5°C, 62°C, 62.5°C, 63°C, 63.5°C, 64°C, 64.5°C, 65°C, 65.5°C, 66°C, 66.5°C, 67°C, 67.5° C, 68°C, 68.5°C, 69°C, 69.5°C, 69.5°C, 70°C, 75°C or more.

[0074] According to particular embodiments, the isothermal amplification is achieved at a temperature ranging from about 60°C to about 70°C.

[0075] According to particular embodiments, the isothermal amplification is selected from the group consisting of Nicking and Extension Amplification Reaction (NEAR), Rolling Circle Amplification (RCA), Helicase-Dependent Amplification (HAD), Loop- Mediated Amplification (LAMP), Transcription-Mediated Amplification (TMA), Self- Sustained Sequence Replication (3SR), Nucleic Acid Sequence Based Amplification (NASBA), Single Primer Isothermal Amplification (SPIA), Q-|3 Replicase System, Strand Displacement Amplification (SDA), Multiple Displacement Amplification (MDA), and Recombinase Polymerase Amplification (RPA).

[0076] According to more particular embodiments, the isothermal amplification is Loop- Mediated Amplification (LAMP).

[0077] Hence, according to more particular embodiments, the isothermal amplification is a Loop-Mediated Amplification (LAMP) that may be carried out at a constanttemperature of about 50°C, 55°C, 60°C, 60.5°C, 61°C, 61.5°C, 62°C, 62.5°C, 63°C, 63.5°C, 64°C, 64.5°C, 65°C, 65.5°C, 66°C, 66.5°C, 67°C, 67.5° C, 68°C, 68.5°C, 69°C, 69.5°C, 69.5°C, 70°C, 75°C or more. According to even more particular embodiments, the Loop-Mediated Amplification (LAMP) is achieved at a temperature ranging from about 60°C to about 70°C.

[0078] Illustratively, the LAMP amplification reaction may be carried out for a period of at least about 2 minutes, at least about 5 minutes, at least about 10 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes or more. In some embodiments, the LAMP amplification reaction may be carried out for a period ranging from about 10 minutes to about 60 minutes, preferably from about 15 minutes to about 45 minutes. Illustratively, the LAMP amplification reaction may be carried out using an isothermal amplification system. Several such instruments are available on the market, and accessible to those skilled in the art.

[0079] According to particular embodiments, the non-human animal sample for which the method is performed, in particular the sample on which a modulation of fluorescence intensity is determined, is further characterized in that it comprises one or more reagent selected from the group consisting of: a cleaning reagent, an extraction reagent, or a combination thereof. In a non-limitative manner, cleaning reagents or extraction reagents may be selected from the group of: anionic detergents, cationic detergents, non-ionic detergents, zwitterionic reagents, and combinations thereof.

[0080] Cleaning reagents / formulations can contain one or more detergents (e.g. surfactants, or combinations thereof). Such surfactants are well known to those skilled in the detergency art. Non limiting examples of possible surfactants include isoceteth-20, sodium methyl cocoyl taurate, sodium methyl oleoyl taurate, and sodium lauryl sulfate. In particular, examples of surfactants and / or detergents may include those selected from the group consisting of: CHAPS, cholic acid, deoxycholic acid, digitonin, n-dodecyl-P- D-maltoside, lauryl sulfate, glycodeoxycholic acid, n-lauroylsarcosine, saponin, triton X- 100, and Sodium Dodecyl Sulfate (SDS).

[0081] According to particular embodiments, the non-human animal sample for which the method is performed, in particular the sample on which a modulation of fluorescence intensity is determined, is characterized in that it comprises at least one detergent; for example an anionic detergent such as Sodium Dodecyl Sulfate (SDS) or a derivative thereof.

[0082] Hence, according to particular embodiments, the non-human animal sample for which the method is performed, in particular the sample on which a modulation of fluorescence intensity is determined, is characterized in that it comprises at least one detergent, for example an anionic detergent, in a concentration of said detergent of 0.01% or more and / or of less than 10.0%, for example ranging from about 0.01 % to about 10.0%.

[0083] For example, the non-human animal sample may comprise at least one detergent, for example an anionic detergent, in a concentration of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%, 2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 4.2%, 4.4%, 4.6%, 4.8%, 5.0%, 5.2%, 5.4%, 5.6%, 5.8%, 6.0%, 6.2%, 6.4%, 6.6%, 6.8%, 7.0%, 7.2%, 7.4%, 7.6%, 7.8%, 8.0%, 8.2%, 8.4%, 8.8%, 9.0%, 9.2%, 9.4%, 9.6%, 9.8%, 10.0%, or more than 10.0%.

[0084] For example, the non-human animal sample may comprise at least one detergent, for example at least one anionic detergent, in a concentration ranging from about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.4%, 1.6%, 1.8%, 2.0%, 2.2%, 2.4%, 2.6%,2.8%, 3.0%, 3.2%, 3.4%, 3.6%, 3.8%, 4.0%, 4.2%, 4.4%, 4.6%, 4.8%, 5.0%, 5.2%, 5.4%,5.6%, 5.8%, 6.0%, 6.2%, 6.4%, 6.6%, 6.8%, 7.0%, 7.2%, 7.4%, 7.6%, 7.8%, 8.0%, 8.2%,8.4%, 8.8%, 9.0%, 9.2%, 9.4%, 9.6%, 9.8%, to about 10.0%, or more than 10.0%.

[0085] According to other particular and non-mutually exclusive embodiments, the non- human animal sample for which the method is performed, in particular the sample on which a modulation of fluorescence intensity is determined, is characterized in that itcomprises at least one salt selected from the group consisting of: a zinc salt, a copper salt, a potassium salt, and combinations thereof.

[0086] According to particular embodiments, the method for detecting nucleic acids and / or for discriminating true-positive isothermal amplification from false-positive isothermal amplification comprises a step of extracting a nucleic acid fraction from the nasal, oral or oropharyngeal sample susceptible to contain the nucleic acid of interest.

[0087] According to more particular embodiments, the extraction of a nucleic acid fraction comprises steps of: al) contacting the nasal, oral or oropharyngeal cat sample susceptible to contain a nucleic acid of interest with a lysis buffer comprising a concentration of SDS ranging from about 1% to about 25%, so that the final concentration of SDS in the reaction mixture is ranging from about 0.01 % to about 10.0%; a2) optionally contacting the mixture from step al) with a buffer comprising a concentration of a zinc and / or copper salt ranging from about 0.5 M to about 5.0 M, so that the final salt concentration of zinc and / or copper in the reaction mixture is ranging from about 10 mM to about 70 mM; a3) contacting the mixture of step al) or a2) with a buffer comprising a concentration of a potassium salt ranging from about 0.1 M to about 5.0 M, so that the final concentration of potassium salt in the reaction mixture is ranging from about 10 mM to about 500 mM.

[0088] For example, the zinc salt may be selected from a group consisting of: zinc sulfate (ZnSCh), and zinc chloride (ZnCh).The copper salt may be selected from a group consisting of: copper sulfate (CuSCh), and copper chloride (CuCh The potassium salt may be selected from the group consisting of potassium hydrogen carbonate (KHCO3), potassium acetate (CH3CO2K), dipotassium hydrogen phosphate (K2HPO4), monobasic potassium phosphate (KH2PO4), and potassium chloride (KC1).

[0089] Optionally, the lysis buffer and / or the buffer comprising a zinc and / or copper salt may comprise one or more additional compound(s) selected in the group consisting of Tris-HCl, HEPES, and MOPS.

[0090] For example, the final concentration of SDS in the reaction mixture at step al) may range from about 1 % to about 10.0%, for example 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.

[0091] For example, the final salt concentration of zinc and / or copper in the reaction mixture may range from about 10, 20, 30, 40, 50, 60 mM to about 70 mM. For example, the final salt concentration of potassium salt in the reaction mixture may range from about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400 mM to about 500 mM.

[0092] In some embodiments, the final concentration of SDS in the reaction mixture is ranging from about 0.06% to about 10%, preferably from about 0.1% to about 6.7%, when the sample (e.g. the complex sample) is selected from group consisting of: a saliva sample, a respiratory lavage sample, and a nasopharyngeal secretion sample.

[0093] In certain embodiments, the final concentration of potassium salt in the reaction mixture is ranging from about 60 mM to about 300 mM, preferably from about 100 mM to about 270 mM, when the sample (e.g. the complex sample) is selected from the group consisting of: a saliva sample, respiratory lavage sample, and a nasopharyngeal secretion sample.

[0094] According to more particular embodiments, the extraction of a nucleic acid fraction, as defined above, further comprises a step a4) of filtering the mixture from step a3), and collecting soluble nucleic acids.

[0095] According to particular embodiments, the method for detecting nucleic acids and / or for discriminating true-positive isothermal amplification from false-positive isothermal amplification is characterized in that the nucleic acid of interest is indicative of an animal infection, in particular a viral infection or a bacterial infection, or a parasitic infection; for example an animal infection for which the pathogen selected from the group consisting of: Feline herpesvirus, Feline Calicivirus, Chlamydia felis. EquineHerpesvirus-1 (EHV-1), Equine Herpesvirus-4 (EHV-4), Influenza, and Streptococcus equi.

[0096] In some embodiments, a bacterium pathogenic for a non-human animal is selected from the non-exhaustive list comprising a bacterium of the species Afipia felis,' a bacterium of the genus Anaplasma, preferably a bacterium of the species Anaplasma phagocy tophilum, Anaplasma cenlrale, Anaplasma mesaelerum, Anaplasma platys, Anaplasma bovis, or Anaplasma o\’is, a bacterium of the genus Bacillus, preferably a bacterium of the species Bacillus anthracis or Bacillus cereus,' a bacterium of the genus Bartonella, preferably a bacterium of the species Bartonella henselae, or Bartonella clarridgeiae,' a bacterium of the genus Bordetella, preferably a bacterium of the species Bordetella pertussis, Bordetella parapertussis or Bordetella bronchiseptica, a bacterium of the genus Borrelia, preferably a bacterium of the species Borrelia burgdorferi, Borrelia recurrentis, Borrelia hispanica, Borrelia persica, Borrelia duttonii or Borrelia crocidurae,' a bacterium of the genus Brucella, preferably a bacterium of the species Brucella melitensis, Brucella abortus or Brucella suis,' a bacterium of the genus Burkholderia, preferably a bacterium of the species Burkholderia mallei or Burkholderia pseudomailer, a bacterium of the genus Campylobacter, preferably a bacterium of the species Campylobacter fetus or Campylobacter jejuni,' a bacterium of the genus Chlamydia, preferably a bacterium of the species Chlamydia trachomatis,' a bacterium of the genus Chlamydophila, preferably a bacterium of the species Chlamydophila pneumoniae or Chlamydophila psittaci,' a bacterium of the genus Clostridium, preferably a bacterium of the species Clostridium botulinum, Clostridium difficile, Clostridium perfringens or Clostridium tetani,' a bacterium of the genus Corynebacterium, preferably a bacterium of the species Corynebacterium diphteriae,' a bacterium of the genus Coxiella, preferably a bacterium of the species Coxiella burnetii,' a bacterium of the genus Ehrlichia, preferably a bacterium of the species Ehrlichia chaffeensis, Ehrlichia equi or Ehrlichia phagocytophila, a bacterium of the genus Erysipelothrix, preferably a bacterium of the species Erysipelothrix rhusiopathiae,' a bacterium of the genus Escherichia, preferably a bacterium of the species Escherichia coli a bacterium of the genus Francisella, preferably a bacterium of the species Francisella tularensis,' a bacterium of the genus Haemophilus, preferably a bacterium of the species Haemophilusducreyi or Haemophilus influenzae,' a bacterium of the genus Helicobacter, preferably a bacterium of the species Helicobacter pylori,' a bacterium of the genus Legionella, preferably a bacterium of the species Legionella pneumophila, a bacterium of the genus Leptospira, preferably a bacterium of the species Leptospira interrogans,' a bacterium of the genus Listeria, preferably a bacterium of the species Listeria monocytogenes,' a bacterium of the genus Mycobacterium, preferably a bacterium of the species Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium bovis or Mycobacterium intracellulare,' a bacterium of the genus Mycoplasma, preferably a bacterium of the species Mycoplasma pneumoniae or Mycoplasma hominis,' a bacterium of the genus Neisseria, preferably a bacterium of the species Neisseria gonorrhoeae or Neisseria meningitidis,' a bacterium of the genus Pasteurella, preferably a bacterium of the species Pasteurella multocida, a bacterium of the genus Pseudomonas, preferably a bacterium of the species Pseudomonas aeruginosa, a bacterium of the genus Rickettsia, a bacterium of the genus Salmonella, preferably a bacterium of the species Salmonella enterica, a bacterium of the genus Shigella, preferably a bacterium of the species Shigella dysenteriae, Shigella jlexneri, Shigella boydii or Shigella sonnei,' a bacterium of the genus Spirillum, preferably a bacterium of the species Spirillum minus,' a bacterium of the genus Staphylococcus, preferably a bacterium of the species Staphylococcus aureus,' a bacterium of the genus Streptococcus, preferably a bacterium of the species Streptococcus pneumoniae,' a bacterium of the genus Treponema, preferably a bacterium of the species Treponema pallidum,' a bacterium of the genus Tropheryma, preferably a bacterium of the species Tropheryma whippier, a bacterium of the genus Ureaplasma, preferably a bacterium of the species Ureaplasma urealyticum,' a bacterium of the genus Vibrio, preferably a bacterium of the species Vibrio cholerae,' a bacterium of the genus Yersinia, preferably a bacterium of the species Yersinia pestis, Yersinia enter ocolitica, or Yersinia pseudotuberculosis.

[0097] In some embodiments, a fungus pathogenic for a non-human animal is selected from the list consisting of, a fungus of the genus Aspergillus, a fungus of the genus Candida.

[0098] In certain embodiments, a protozoan pathogenic for a non-human animal is selected from the list consisting of, a protozoan of the genus Babesia, a protozoan of the genus Plasmodium, a protozoan of the genus Theileria, a protozoan of the genus Toxoplasma. The virus can be a DNA virus or an RNA virus.

[0099] In some embodiments, the DNA virus is selected from the non-exhaustive list comprising a virus of the family Adenoviridae, preferably a virus of the genus Adenovirus,' of the Hepdnaviridae family, preferably a virus of the genus Herpadnavirus,' from the family of Herpesviridae, preferably a virus of the genus Herpesvirus,' of the Papovaviridae family, preferably a virus of the genus Polyomavirus or Papillomavirus,' of the Parvoviridae family, preferably a virus of the Parvovirus genus; of the Poxviridae family, preferably a virus of the Poxvirus genus. In some embodiments, the DNA virus may be a double stranded (ds) DNA virus, in particular a virus selected in a group consisting of a virus of the Hepadnaviridae family, Circoviridae family, Herpesviridae family, Poxviridae family, Adenoviridae family, Papillomaviridae family, and Polyomaviridae family. In some embodiments, the DNA virus may be a single stranded (ss) DNA virus, including a virus of the Parvoviridae family. In some embodiments, the RNA virus is selected from the list consisting of a virus of the Arenaviridae family, preferably a virus of the Arenavirus genus; of the Bunyaviridae family, preferably a virus of the Bunyavirus, Phlebovirus, Nairovirus or Hantavirus genus; of the Caliciviridae family, preferably a virus of the Calicivirus genus; of the Coronaviridae family, preferably a virus of the Coronavirus genus; from the Filoviridae family, preferably a virus from the Filovirus genus; of the Flaviviridae family, preferably a virus of the Flavivirus, Ribivirus or Hepcivirus genus; from the Orthomyxoviridae family, preferably a virus of the genus Influenzavirus,' of the Paramyxoviridae family, preferably a virus of the Pneumovirus, Paramyxovirus or Morbillivirus genus; of the Picornaviridae family, preferably a virus of the Enterovirus, Hepatovirus or Rhinovirus genus; of the Rhabdoviridae family, preferably a virus of the genus Lyssavirus or Vesiculovirus,' of the Reoviridae family, preferably a virus of the Reovirus or Rotavirus genus; of the Retroviridae family, preferably a virus of the genus Oncornavirus or Lentivirus,' from the Togaviridae family, preferably a virus from the genus Alphavirus. In some embodiments, the RNA virus may be a double stranded RNA virus, including a virus of the Reoviridaefamily, in particular a rotavirus, such as, the bluetongue virus. In some embodiments, the RNA virus may be a negative-sense, single strand (ss)(-)RNA virus, in particular selected in a group consisting of a virus of the Orthomyxoviridae family, Filoviridae family, Paramyxoviridae family, Rhabdoviridae family, Arenaviridae family, Bunyaviridae family, Qinviridae family, Aspviridae family, and Yueviridae family. In some embodiments, the RNA virus is a positive-sense, single strand ss(+)RNA virus, in particular selected in a group consisting of a virus of the Coronaviridae family, including SARS-CoV-2, SARS-CoV and MERS-CoV; a virus of the Picornaviridae family, including Poliovirus, Rhinovirus, Aphthovirus, Cardiovirus, Coxsackie Viruses, and Hepatitis A virus; a virus of the Calciviridae family, including Norwalk virus and Hepatitis E virus; a virus of the Flaviviridae family, including Yellow fever virus, West Nile Virus, Hepatitis C virus, Dengue virus and Zika virus; a virus of the Togaviridae family, including rubella virus and Chikungunya virus. In some embodiments, the virus of the Coronavirus genus may be a SARS-CoV-2 virus, a SARS- CoV virus, a MERS-CoV virus. In some embodiments, the virus is selected from a list consisting of, African horse sickness virus, African swine fever virus, Andes virus, avian influenza virus, ovine bluetongue virus, Chapare virus, Chikungunya virus, Choclo virus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dobrava-Belgrade virus, Eastern equine encephalitis virus, Ebola virus, foot-and-mouth disease virus, goat pox virus, Guanarito virus, human immunodeficiency virus (HIV), Hantaan virus, Hendra virus, porcine herpesvirus (or Aujeszky's disease), swine fever virus, Japanese encephalitis virus, Junin virus, Kyasanur forest disease virus, Laguna negra virus, Lassa fever virus, ovine encephalomyelitis virus, Lujo virus, lumpy skin disease virus, Lymphocytic choriomeningitis virus, Machupo virus, Marburg virus, MERS-CoV virus, Monkey pox virus, Murray Valley encephalitis virus, Newcastle disease virus, Nipah virus, Omsk Haemorrhagic fever virus, Oropouche virus, small rodents plague virus, porcine type 9 enterovirus (or swine vesicular disease virus), Powassan encephalitis virus, rabies virus, Rift Valley virus, Rinderpest virus, Rocio virus, Sabia virus, SARS-CoV virus, SARS- CoV-2 virus, Seoul virus, Sheep pox virus, Sin Nombre virus, St. Louis encephalitis virus, Teschen's disease, tick-borne encephalitis virus, smallpox virus, Venezuelan equine encephalitis virus, vesicular stomatitis virus, western equine encephalitis virus, and yellow fever virus.

[0100] According to particular embodiment, the nucleic acid of interest may thus be indicative of a cat infection ; in particular a cat viral infection or a cat bacterial infection, or a cat parasitic infection; for example selected from the group consisting of: Feline herpesvirus, Feline Calicivirus, Chlamydia felis, or any variant thereof.

[0101] According to a particular embodiment, the nucleic acid of interest may thus be indicative of an equine infection ; in particular an equine viral infection or an equine bacterial infection, or an equine parasitic infection; for example selected from the group consisting of Equine Herpesvirus- 1 (EHV-1), Equine Herpesvirus-4 (EHV-4), Influenza, and Streptococcus equi, or any variant thereof

[0102] Nucleic acids being indicative of an animal parasitic infection may further include those from parasites belonging to the phylum Apicomplexa, such as those belonging to the genus Theileria, and those belonging to the genus Babesia ; for example those selected from the group consisting of : Theileria equi, Theileria annulata, Theileria electrophori, Theileria orientalis Theileria parva and Theileria microti, Babesia cavalli, Babesia divergens, Babesia microti, Babesia duncani et Babesia venatorum, or any variant thereof.

[0103] Nucleic acids being indicative of an animal bacterium infection may further include those from bacteria belonging to the phylum spirochaeota, such as those belonging to the genus Leptospira , ' for example those selected from the group consisting of : Leptospira interrogans, Leptospira borgpetersenii, Leptospira kirschneri, Leptospira alstoni, Leptospira noguchii, Leptospira alexanderi, Leptospira weilii, Leptospira santarosai, Leptospira kmetyi, Leptospira mayottensis, Leptospira inadai, Leptospira fainei, Leptospira broomii et Leptospira wolffii, or any variant thereof.

[0104] Nucleic acids being indicative of an animal bacterium infection may further include those from bacteria belonging to the genus Anaplasma or Borrelia, for example those selected from the group consisting of : Anaplasma phagocy tophilum, B garinii, B. bavariensis, B. spielmanii, B. afzelli, B. burgdorferi., hispanica, B. recurrentis, B. persica, B. duttonii, B. crocidurae, B. turicatae, B. parkeri, B. hermsii, B. miamotoi et B. venezuelensis, or any variant thereof

[0105] According to a third main embodiment, the invention relates to an in vitro use of melting curve analysis for discriminating true-positive isothermal amplification from false-positive isothermal amplification in a nasal, oral or oropharyngeal non-human animal sample.

[0106] Hence, according to said main embodiment, the invention may relate to an in vitro use of melting curve analysis for discriminating true-positive isothermal amplification from false-positive isothermal amplification in a nasal, oral or oropharyngeal non-human cat or equine sample.

[0107] It will be readily understood by the reader that melting curve analysis may be achieved as described previously.

[0108] Advantageously, the in vitro use of melting curve analysis, the in vitro method for detecting nucleic acids in a non-human animal sample, or for discriminating truepositive isothermal amplification from false-positive isothermal amplification in a non- human animal sample, can be further combined to a method (e.g. an in vitro method) for detecting the occurrence of a disorder in a non-human animal.BRIEF DESCRIPTION OF THE DRAWINGS

[0109] Figure 1. LAMP amplification (1A) and post-amplification melting curve (IB) on four healthy cat samples. (1A) Time is expressed in minutes in the x-axis. Fluorescence is expressed in arbitrary units in the y-axis. (IB) Temperature is expressed in Celsius degrees in the x-axis. Fluorescence is expressed in arbitrary units in the y-axis. Each cat sample (2329, 2330, 2331, 2332) for which nucleic acid amplification is measured with a cat-specific DNA sequence is reported as a plain line. Each cat sample (2329, 2330, 2331, 2332) for which nucleic acid amplification is measured with a Chlamydia felis sequence is reported as a corresponding dotted line.

[0110] Figure 2. LAMP amplification (2A) and post-amplification melting / / annealing curve on an infected cat positive toward Feline Calicivirus, and Chlamydia felis (2B). Figure legends for x-axis and y-axis are as reported for figure 1.Each cat sample (2355) for which nucleic acid amplification is measured with a catspecific DNA sequence is reported as a plain line. Each cat sample for which nucleic acid amplification is measured with a herpesvirus or calicivirus specific sequence is reported with a grey line. The cat sample for which nucleic acid amplification is measured with a Chlamydia felis reference sequence is reported as a dotted line.

[0111] Figure 3. LAMP amplification (3A) and post-amplification melting / annealing curve (3B) on a false-positive sample for Feline Calicivirus. Figure legends for x-axis and y-axis are as reported for figure 1. Each cat sample (2877) for which nucleic acid amplification is measured with a cat-specific DNA sequence is reported as a plain line. Each cat sample for which nucleic acid amplification is measured with a herpesvirus or calicivirus specific sequence is reported with a grey line. The cat sample for which nucleic acid amplification is measured with a Chlamydia felis reference sequence is reported as a dotted line.

[0112] Figure 4. LAMP amplification (4A) and post-amplification melting / annealing curve (4B) on a false-positive sample for Feline Calicivirus with early amplification, and a post-amplification curve indicating positivity. Figure legends for x-axis and y-axis are as reported for figure 1. The cat sample (2867) for which nucleic acid amplification is measured with a cat-specific DNA sequence is reported as a plain line. Each cat sample for which nucleic acid amplification is measured with a herpesvirus or calicivirus specific sequence is reported with a grey line.

[0113] Figure 5. LAMP amplification (5A) and post-amplification melting / annealing curve (5B) on a false-positive horse sample for EHV-1, EHV-4, Influenza, and S.equi. Figure legends for x-axis and y-axis are as reported for figure 1. The horse sample (2634) for which nucleic acid amplification is measured with a horsespecific DNA sequence is reported as a plain line. Amplification with EHV-1 or EHV-4 specific sequence is reported with a grey line. Amplification with Influenza or S.equi specific sequence is reported with a grey line.EXAMPLES

[0114] The present invention is further illustrated by the following examples.

[0115] In this study, the inventors have sought to determine whether post-LAMP amplification melting curves would be sufficient for detecting non-specific amplification in oropharyngeal swabs of cats, in the context of a standardized test for detecting respiratory feline pathogens such as herpesvirus, calicivirus, and chlamydia felis, which are causative pathogens of the feline Coryza syndrome.Materials and Methods

[0116] Feline swabs were taken from 34 different cats by rubbing the tongue and gums. The samples were then received in the laboratory and an “Enalees” extraction was carried out as described in WO2021 / 160849 Al.

[0117] The extracted material is brought into a SDS sample for inducing cell lysis. A KHCO3 composition is added for inducing precipitation of SDS. The resulting composition is filtered and the SDS / KHCO3 is discarded. The final filtrated composition including the extracted nucleic acid preparation corresponds to the DNA to be amplified.

[0118] The extracted material is then combined with a rehydration buffer containing MgSO4 and then mixed with a LAMP mastermix containing the enzyme, a fluorescent intercalating dye (VI 3 -01184 sold by Dyomics), dNTPs, primers specific to the DNA to be amplified, and for test samples including RNA, a reverse transcriptase).

[0119] The extracted material was distributed into each lyophilized well: feline endogenous control, FHV (feline herpesvirus), FCV (feline calicivirus) or Cfelis (Chlamydia felis). All reagents used have been previously validated following quality control. Feline endogenous control (ND5) ensures that the sample has been correctly used and contains enough cat cells to be used for pathogen detection.

[0120] For isothermal amplification on swab samples, an amplification program of 30minutes at 65°C was started, followed by a fusion program of approximately 5 min with a temperature ranging from 95 to 65°C or from 98°C to 60°C or from 92°C to 65°C. For fluorescence measurement, a read is obtained for 10 seconds.

[0121] Post-amplification curves are obtained on either a QuantStudio 5 (QS5) thermocycler (ThermoFisher Scientific) or a Gene-8C isothermal fluorescence PCR (Allsheng).

[0067] The Gene-8C apparatus consists of a heating module (Peltier-type) coupled to a spectrofluorometer, which is specifically adapted to LAMP amplification. The corresponding software for visualizing amplification curves is not capable of derivatizing a fluorescence variation as a function of temperature, but instead provides the fluorescence value as a function of temperature directly.Results test with a QS5 thermocycler forto a true-

[0122] A synthetic double-stranded linear DNA (SYNTH 040) containing the sequence to be amplified (amplified sequence specific for Feline calicivirus) was inserted into a reaction tube. Therefore we can be sure that the observed amplification is a real amplification. LAMP amplification was carried out (standard LAMP amplification curves not shown) then an annealing step was carried out. The following table gives the melting temperature.

[0123] The observed melting temperature (Tm) for the amplification of a reference truepositive sample is measured from 89.3°C to 89.9°C for the corresponding DNA sequence.2: Preliminary test with a QS5 thermocycler for determining melting

[0124] A sample that was identified as negative for Feline Calicivirus by PCR wasinserted into a reaction tube. By carrying out a LAMP reaction, a very early amplification product with the appearance of a classic LAMP amplification curve was observed (LAMP curve not shown) for all the tests carried out (Control, Feline herpesvirus, Feline calicivirus, Chlamydia felis) which suggests a non-specific amplification of the sample. Indeed, it is unlikely that a cat is contaminated by all of these pathogens even though a PCR was negative.

[0125] An annealing step was carried out to see if the melting temperature was the same as in the case of the true positives. The following table gives the melting temperature.

[0126] The observed melting temperature (Tm) for the amplification of the false-positive sample is measured from 69.72°C to 69.87°C for the corresponding DNA sequence.

[0127] As a conclusion, it can be found a difference of about 20°C between the experimental Tm of true-positives and false-positives. Post-:melting curve on a Gene-8C thermocycler forcat swabA. Measurement on a control sample from four healthy cat samples

[0128] The oropharyngeal cat samples (codenamed 2329, 2330, 2331 and 2332) are tested by isothermal amplification of a cat-specific DNA sequence, and a Chlamydia felis reference sequence.

[0129] During the LAMP amplification, only one amplification is detected, corresponding to the amplification of the cat-specific DNA sequence, thus demonstrating that the sample was recovered properly.

[0130] No aspecific amplification is found (i.e. no interference with cat saliva is found). Although no melting / annealing curve is required in this case, the sample is tested as a control experiment, and a Tm of 86°C is found. The outcomes of the LAMP amplification and of the post-amplification melting curve are reported, respectively, in figure 1A andfigure IB.

[0131] No amplicon corresponding to the pathogens is detected; therefore no Tm can be measured for those amplicons.B. Determination of feline calicivirus from an infected cat as positive control).

[0132] The oropharyngeal cat sample (positive control; codenamed 2355) is tested for Feline herpesvirus, Feline Calicivirus, and Chlamydia felis, as previously.

[0133] During the LAMP amplification, three amplifications are detected, corresponding to the amplification of the cat-specific DNA sequence, and the amplification of a DNA sequence specific to Feline calicivirus, and the amplification of a DNA sequence specific to Chlamydia felis.

[0134] No aspecific amplification is found. Although no melting / annealing curve is required in this case, the sample is tested for both the cat-specific DNA sequence and the amplification of a DNA sequence specific to Feline calicivirus as a positive control experiment. A Tm of 88°C is found for the cat-specific DNA sequence, a Tm of 90°C is found for Feline calicivirus and a Tm of 88°C is found for Feline Chlamydia felis. No other amplicon is detected for the other pathogenss. The outcomes of the LAMP amplification and of the post-amplification melting curve are reported, respectively, in figure 2 A and figure 2B.C. Case study on a false-positive sample for Feline Calicivirus with postamplification melting curve indicating negativity

[0135] The oropharyngeal cat sample (codenamed 2877) is tested for Feline herpesvirus, Feline Calicivirus, and Chlamydia felis, as previously.

[0136] During the LAMP amplification, four amplifications are detected, corresponding to the amplification of the cat-specific DNA sequence, and theamplification of a DNA sequence specific to Feline Calicivirus, Feline herpesvirus and Chlamydia felis except that, in this case, the cat sample is reported to be negative toward all the tested pathogens according to Polymerase Chain Reaction (PCR).

[0137] The observed additional amplification thus appears to be linked to the presence of an unidentified compound in the mouth of the cat, at the time of sample recovery. Another reason is the unlikely scenario that the same cat would be contamined by all three pathogens.

[0138] In this case, annealing curves are achieved; thereby providing a Tm of 65°C for cat-specific DNA sequence and a Tm of 65°C for the pathogen sequences. Hence, the measured Tms are not in accordance with the expected Tms; thereby reaching the conclusion that a false-positive is present. The outcomes of the LAMP amplification and of the post-amplification melting curve are reported, respectively, in figure 3A and figure 3BD. Case study on a false-positive sample for Feline Calicivirus with early amplification, and a post-amplification curve indicating positivity.

[0139] The oropharyngeal cat sample is tested for Feline herpesvirus, and Feline Calicivirus.

[0140] During the LAMP amplification, three amplifications are detected, corresponding to the amplification of the cat-specific DNA sequence, and the amplification of a DNA sequence specific to Feline herpesvirus and Feline Calicivirus; except that, in this case, the cat sample is reported to be positive toward Feline herpes virus only, according to Polymerase Chain Reaction (PCR). It is also unlikely that the cat was co-infected by two distinct pathogens.

[0141] In contrast to case study C, the observed amplifications occur at an earlier stage, and they may thus be masked by an aspecic amplification.

[0142] Annealing curves are achieved which provide evidence for a Tm of 86°C for cat-specific DNA sequence and a Tm of 86°C for the Feline herpesvirus sequence. Hence, the measured Tms are in accordance with the expected values; thereby reaching the conclusion that a true-positive is present in the tested cat sample (codenamed 2867).

[0143] On the other hand, annealing curves are achieved which provide evidence for a Tm of 65°C for the Feline calicivirus sequence, which is not in accordance with the expected values. Therefore, the results for Feline calicivirus are false-positives. The outcomes of the LAMP amplification and of the post-amplification melting curve for feline calicivirus are reported, respectively, in figure 4A and figure 4B. E. Case study on a false-positive horse sample for EHV-1, EHV-4, Influenza, and Streptococcus equi.

[0144] The oropharyngeal horse sample is tested for Equine Herpesvirus-1 (EHV-1), Equine Herpesvirus-4 (EHV-4), Influenza, and S.equi. An equine reference nucleic acid sequence is considered for control.

[0145] As stated previously, the LAMP amplification corresponds to a false-positive toward all four pathogens.

[0146] The outcomes of the LAMP amplification and of the post-amplification melting curve for feline calicivirus are reported, respectively, in figure 5A and figure 5B.

Claims

CLAIMS1. An in vitro method for detecting nucleic acids in a non-human animal sample, comprising steps of: a) providing a nasal, oral or oropharyngeal non-human animal sample susceptible to contain a nucleic acid of interest, or a fraction thereof; b) detecting a modulation of fluorescence intensity on or after isothermal amplification of the sample, or fraction thereof, thereby detecting an amplified fraction; c) detecting a modulation of fluorescence intensity as a function of temperature in the amplified fraction, and comparing it to a reference value, thereby detecting the nucleic acid of interest.

2. An in vitro method for discriminating true-positive isothermal amplification from false-positive isothermal amplification in a non-human animal sample, comprising steps of: a) providing a nasal, oral or oropharyngeal non-human animal sample susceptible to contain a nucleic acid of interest, or a fraction thereof; b) detecting a modulation of fluorescence intensity on or after isothermal amplification of the sample, or fraction thereof, thereby detecting an amplified fraction; c) detecting a modulation of fluorescence intensity as a function of temperature in the amplified fraction, and comparing it to a reference value, thereby discriminating true-positive isothermal amplification from false-positive isothermal amplification.

3. The method according to claim 1 or 2, wherein the non-human animal sample is a cat sample.

4. The method according to claim 1 or 2, wherein the non-human animal sample is an equine sample.

5. The method according to any of claims 1 to 4; wherein the non-human animal sample is an oral sample; in particular a swab sample.

6. The method according to any of claims 1 to 5; wherein modulation of fluorescence intensity is detected by measuring a modulation of a fluorescence signal from one or more intercalating dyes and / or fluorescent probes.

7. The method according to any of claims 1 to 6; wherein modulation of fluorescence intensity as a function of temperature in the amplified fraction, at step c), is achieved by modulating the temperature of the amplified fraction in a range from about 60°C to about 98°C.

8. The method according to any of claims 1 to 5, wherein the reference value corresponds to, or is indicative of, a melting temperature corresponding to one or more double-stranded nucleic acids, in particular double-stranded deoxyribonucleic acids (dsDNA), in the amplified fraction.

9. The method according to claim 8, wherein the reference value is determined on a reference sample comprising the nucleic acid of interest.

10. The method according to any of claims 1 to 9; wherein isothermal amplification is achieved at a temperature ranging from about 60°C to about 70°C.

11. The method according to any of claims 1 to 10; wherein isothermal amplification is selected from the group consisting of: Nicking and Extension Amplification Reaction (NEAR), Rolling Circle Amplification (RCA), Helicase- Dependent Amplification (HAD), Loop-Mediated Amplification (LAMP), Transcription-Mediated Amplification (TMA), Self-Sustained Sequence Replication (3SR), Nucleic Acid Sequence Based Amplification (NASBA), Single Primer Isothermal Amplification (SPIA), Q-P Replicase System, Strand Displacement Amplification (SDA), Multiple Displacement Amplification (MDA), and Recombinase Polymerase Amplification (RPA).

12. The method according to claim 11; wherein isothermal amplification is Loop-Mediated Amplification (LAMP).

13. The method according to any of claims 1 to 12; wherein the method comprises a step of extracting a nucleic acid fraction from the nasal, oral or oropharyngeal sample susceptible to contain the nucleic acid of interest.

14. The method according to any of claims 1 to 13, wherein the nucleic acid of interest is indicative of an animal infection, in particular a viral infection or a bacterial infection, or a parasitic infection; for example selected from the group consisting of Feline herpesvirus, Feline Calicivirus, Chlamydia felis. Equine Herpesvirus-1 (EHV-1), Equine Herpesvirus-4 (EHV-4), Influenza, and Streptococcus equi.

15. In vitro use of melting curve analysis for discriminating true-positive isothermal amplification from false-positive isothermal amplification in a nasal, oral or oropharyngeal non-human animal sample, or a fraction thereof.

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