KITS AND METHODS FOR EXTRACTION OF NUCLEIC ACIDS FROM COMPLEX SAMPLES

Abstract

KITS AND METHODS FOR EXTRACTION OF NUCLEIC ACIDS FROM COMPLEX SAMPLES The present invention relates to a kit and a method for the extraction of nucleic acids from complex samples. More particularly, the kit comprises: (i) a lysis buffer comprising an SDS concentration of between approximately 1% and 25%; (ii) a buffer comprising a potassium salt concentration of between approximately 10 mM and 50 mM; (iii) a buffer comprising a zinc salt concentration of between approximately 0.5 M and 5.0 M; (iv) a filter having a pore diameter of between approximately 1 µm and 10 µm; and optionally, an element selected from a syringe for use with the filter, one or more reaction tubes, an instruction guide, and a combination thereof.

Description

Title of the invention: KITS AND METHODS FOR THE EXTRACTION OF NUCLEIC ACIDS FROM COMPLEX SAMPLES FIELD OF THE INVENTION

[0001] The present invention relates to the field of nucleic acid extraction. More particularly, the invention relates to a kit and a method for extracting nucleic acids from complex samples, quickly and economically, without the need for tedious purification steps and without requiring sophisticated equipment and suitable dedicated premises. STATE OF THE ART

[0002] The characterization of nucleic acids from a sample from a particular environment makes it possible to obtain a large amount of information on the nature of this environment. For example, advances in high-throughput sequencing have recently made it possible to make the diagnosis of genetic or infectious diseases more efficient. Indeed, precise knowledge of genetic information makes it possible to provide a targeted therapeutic approach. These advances also make it possible to identify the species populating a given biotope with great efficiency.

[0003] As a general rule, nucleic acids are contained in cells, viruses, parasites, and are therefore protected from external attacks by more or less sophisticated and robust membrane or wall systems. Thus, current nucleic acid analysis techniques necessarily require a first extraction step, i.e. the separation of the nucleic acids from the other cellular components. In practice, this involves lysing the membranes or walls to release the nucleic acids, which can then be purified from the other cellular components and / or other components of the original environment.

[0004] Several nucleic acid extraction methods have been developed in the past, involving both physical lysis processes, such as pressure or ultrasound, and chemical lysis processes. A frequently used chemical lysis method is based on the use of sodium dodecyl sulfate (or SDS), a strong ionic detergent and surfactant, which is generally combined with proteases for even more efficient lysis. This chemical method based on the use of SDS allows the rupture of membranes or walls and the release of nucleic acids. For example, extraction methods based on SDS lysis are used in various fields, such as the study of seabed biomass (Natarajan et al.; Front. Microbiol., 2016, 7:986), or DNA extraction bacterial plasmid (the "miniprep" principle; see, for example, Green and Sambrook (Molecular Cloning: A Laboratory Manual; 4th edition; Cold Spring Harbor Laboratory Press; 2012). However, the presence of SDS often poses a problem during subsequent steps of nucleic acid analysis. SDS is particularly known to be an inhibitor of PCR ("Polymerase Chain Reaction") amplification. Thus, extraction methods specifically compatible with subsequent PCR steps have been developed (see, for example, Goldenberger et al.; Genome Res., 1995, 4:368-370).

[0005] In practice, SDS-based extraction methods almost always include nucleic acid purification steps. Purification can be carried out on affinity or exclusion columns, by using a phenol / chloroform mixture or other chemicals. Alternatively or simultaneously, chemical compounds such as acetamide, betaine, bovine serum albumin, dextran, DMSO, Tween, etc., can be incorporated into the reaction mixture during lysis if a subsequent PCR amplification step is envisaged (see e.g. Hedman and Radström; PCR Detection of Microbial Pathogens: Second Edition, Methods in Molecular Biology, vol. 943, Chapter 2; Abu Al-Soud and Radström; Journal of Clinical Microbiology, 2000, Vol. 38, No. 12, pp. 4463-4470).

[0006] These methods therefore have the disadvantages of being long, tedious, of using chemical compounds which are often toxic for the operator and for the environment, of requiring expensive equipment, such as extractor hoods, protective gloves, centrifuges, etc., and of being implemented in suitable premises, such as laboratories.

[0007] However, there are many situations in which rapid extraction, not involving long and laborious purification steps and not necessarily being carried out in a laboratory setting, is advantageous. As a particularly illustrative example, the case of the veterinarian wishing to quickly identify an infectious pathogen directly at the location of the sick animal can be cited. This approach, known as "point-of-care testing" (POCT), is the subject of particular attention among the professionals concerned.

[0008] Furthermore, it appears that depending on the nature of the original sample, the extraction protocols must be adapted. For example, specific nucleic acid extraction kits for blood samples and for fecal samples have been developed on the market (see for example the Qiagen® kit for the extraction of nucleic acid from a blood sample (QIAamp® DNA Mini and Blood Mini) or from a fecal sample (QIAamp® Fast DNA Stool Mini)). It should be noted that these kits make it possible to obtain nucleic acids of a high degree of purity, compatible with most of the available analysis techniques, in approximately 20 to 30 minutes.

[0009] There is therefore a need to provide means allowing rapid, inexpensive extraction of nucleic acids, which is not harmful to the operator or the environment, and which does not require the constraint of implementation in a dedicated laboratory.

[0010] There is also a need to provide means allowing “universal” nucleic acid extraction, regardless of the nature of the original sample containing the nucleic acids. SUMMARY

[0011] A first aspect of the invention relates to a kit for extracting nucleic acids contained in a complex sample, said kit comprises:

[0012] - a lysis buffer comprising an SDS concentration of between approximately 1% and 25%, preferably between approximately 5% and 20%; - a buffer comprising a concentration of a potassium salt of between approximately 10 mM and 500 mM, preferably of between approximately 60 mM and 400 mM; - a buffer comprising a concentration of a zinc salt of between approximately 0.5 M and 5.0 M; - a filter having a pore diameter of between approximately 1 pm and 10 pm, preferably between approximately 2 pm and 7 pm; - optionally, one element selected from a syringe intended for use with the filter, one or more reaction tubes, an instruction guide, and their combination.

[0013] In some embodiments, the potassium salt is potassium hydrogen carbonate (CHKO3) and / or the zinc salt is zinc sulfate (ZnSO4).

[0014] The invention also relates to a use of a kit according to the present invention, in a method for extracting nucleic acids contained in a complex sample.

[0015] In another aspect, the invention relates to a method for extracting nucleic acids contained in a complex sample, said method comprising the following steps:

[0016] - a) contacting the complex sample with a lysis buffer comprising an SDS concentration of between approximately 1% and 25%, so that the final SDS concentration in the reaction mixture is between approximately 0.01% and 10%; - b) optionally bringing the mixture from step a) into contact with a buffer comprising a concentration of a zinc salt of between approximately 0.5 M and 5.0 M, such that the final zinc salt concentration in the reaction mixture is between about 10 mM and 70 mM; - c) contacting the mixture from step a) or b) with a buffer comprising a concentration of a potassium salt of between approximately 0.1 M and 5.0 M, so that the final concentration of potassium salt in the reaction mixture is between approximately 10 mM and 500 mM; - d) filtering the reaction mixture from step c) so as to collect soluble nucleic acids.

[0017] In some embodiments, the complex sample is selected from the group comprising a biological, environmental, agri-food sample, preferably a biological sample. In some embodiments, step b) is carried out when the complex sample is a blood sample. In some embodiments, the final concentration of SDS in the reaction mixture is between about 0.01% and 1%, preferably between about 0.25% and 0.75%, when the complex sample is a blood sample. In some embodiments, the final concentration of potassium salt in the reaction mixture is between about 10 mM and 500 mM, preferably between about 25 mM and 150 mM, when the complex sample is a blood sample.In some embodiments, the final SDS concentration in the reaction mixture is between about 0.06% and 10%, preferably between about 0.1% and 6.7%, when the complex sample is selected from the group consisting of a fecal sample, a saliva sample, and a nasopharyngeal secretion sample. In some embodiments, the final potassium salt concentration in the reaction mixture is between about 60 mM and 300 mM, preferably between about 100 mM and 270 mM, when the complex sample is selected from the group consisting of a fecal sample, a saliva sample, and a nasopharyngeal secretion sample. DEFINITIONS

[0018] In the present invention, the terms below are defined as follows:

[0019] - The term “Approximately”, placed before a number, means plus or minus 10% of the nominal value of this number. - The term “Nucleic acid extraction” refers to the action of physically separating nucleic acids from other constituents of the biological entity that contains them. Since nucleic acids are contained in biological entities and protected from the environment by walls, nucleic acid extraction generally includes a step of solubilization of the walls and the release of the nucleic acids into the reaction medium; these phenomena are also defined as lysis. - The term “Sample” refers to a biological material obtained, preferably previously obtained by an appropriate sampling technique from an individual, in an environment, or from an agri-food product. By extension, “Complex Sample” refers to a sample which may include, in addition to the nucleic acids of interest, other constituents, such as polypeptides, lipids, polysaccharides, salts, metals, trace elements, solvents, acids, bases. - The term “Buffer” refers to a solution that allows its original pH to be maintained despite the limited addition of an acid or a base or despite dilution. - The term “Reaction mixture” relates to an environment in which a reaction occurs. For example, biological entities contained in a sample and comprising nucleic acids of interest may be lysed upon contact with a buffer comprising SDS. In the context of the invention, “Reaction mixture” means the mixture of all the ingredients used by the method of the invention, and includes the sample containing the nucleic acids of interest, the lysis buffer, the buffer comprising the potassium salt and optionally the buffer comprising the zinc salt. - The term “Amplification” concerns the targeted multiplication of the number of copies of the nucleic acids of interest. - The term “Diagnosis” relates to both medical diagnosis and veterinary diagnosis and relates to the identification of the nature of the pathology in a sick individual. According to one embodiment, the diagnostic methods described herein are non-invasive, i.e., in vitro, diagnostic methods. In this embodiment, a sample to be analyzed is previously taken from the subject before implementing the methods of the invention. - The term “Individual” refers to an animal, vertebrate or invertebrate, preferably a mammal. The mammal may be a non-human mammalian animal or a human. DETAILED DESCRIPTION

[0020] The inventors observed that it was possible to extract nucleic acids contained in complex samples, and intended for analysis by amplification, quickly and inexpensively, without additional purification steps being necessary. Furthermore, the method according to the invention does not require the use of compounds intended to trap amplification inhibitors likely to be contained in complex samples. This method of extracting nucleic acids cleiques is thus compatible with the search for an infection by a pathogenic microorganism in a sick individual, and the subsequent diagnosis.

[0021] The present invention relates to a kit for extracting nucleic acids contained in a complex sample, said kit comprises:

[0022] - a lysis buffer comprising an SDS concentration of between approximately 1% and 25%, preferably between approximately 5% and 20%; - a buffer comprising a concentration of a potassium salt of between approximately 10 mM and 500 mM, preferably of between approximately 60 mM and 400 mM; - a buffer comprising a concentration of a zinc salt of between approximately 0.5 M and 5.0 M; - a filter having a pore diameter of between approximately 1 pm and 10 pm, preferably between approximately 2 pm and 7 pm; - optionally, one element selected from a syringe intended for use with the filter, one or more reaction tubes, an instruction guide, and their combination.

[0023] In the context of the invention, an SDS concentration of between 1% and 25% is understood as an SDS concentration of between 1 g / 100 ml and 25 g / 100 ml.

[0024] In practice, the buffers in the kit are contained in dedicated receptacles, in particular bottles or tubes. The receptacles may be made of a suitable material, such as, e.g., polypropylene, acrylic, polyvinyl chloride.

[0025] In practice, the nucleic acids concerned by the invention are deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), including messenger ribonucleic acids (mRNAs), transfer ribonucleic acids (tRNAs), ribosomal ribonucleic acids (rsRNAs).

[0026] In one embodiment, the nucleic acids are exclusively deoxyribonucleic acids (DNAs). In one embodiment, the nucleic acids are exclusively ribonucleic acids (RNAs). In one embodiment, the nucleic acids are a mixture of deoxyribonucleic acids (DNAs) and ribonucleic acids (DNAs).

[0027] In practice, the sample can be of a very varied nature. Illustratively, the complex sample can be of biological, environmental or agri-food origin.

[0028] In one embodiment, the sample is a biological sample, in particular a biological fluid or a biopsy. In some embodiments, the sample is a sample of a biological fluid, for example selected from the non-exhaustive list comprising, bile, feces, aqueous humor, milk, amniotic fluid, lymph, cerebrospinal fluid, plasma, the product of a lavage res sputum, guttural pouch product, pus, nasopharyngeal secretion, lacrimal secretion, vaginal secretion, saliva, blood, serum, semen, and urine. In some embodiments, the sample is a biopsy, for example selected from the non-exhaustive list including a liver biopsy, an intestinal biopsy, a muscle biopsy, a bone biopsy, a pancreas biopsy, a skin biopsy, a lung tissue biopsy, a vascular tissue biopsy, a bladder biopsy.

[0029] In some embodiments, the sample is an environmental sample such as seawater, freshwater, wastewater, ice, mud, soil.

[0030] As defined in the context of the invention, the term “waste water” refers to water which has been used domestically, agriculturally or industrially and circulates in a sanitation network.

[0031] In certain embodiments, the sample is an agri-food sample intended for animal or human consumption and containing an ingredient such as a cereal, e.g., wheat, barley, oats; a legume, e.g., lentils; a meat, e.g., beef, veal, mutton, lamb, pork, rabbit, poultry; a fish, e.g., cod, trout, salmon; seafood, e.g., oysters, mussels, shrimp, lobster; a vegetable, e.g., apricot, eggplant, banana, carrot, cherry, cabbage, zucchini, strawberry, green bean, kiwi, lettuce, pear, apple, potato. In some embodiments, the agri-food sample may be derived from a manufactured food product, such as, e.g., a ready meal, a can of food, a frozen product.

[0032] In some embodiments, the sample is a sample likely to contain animal and / or plant cells.

[0033] In one embodiment, the animal cell is selected from the non-exhaustive list comprising 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 polymorphonuclear cell; a hepatocyte; a keratinocyte; a nerve cell; and their combination.

[0034] In some embodiments, the sample is a sample likely to contain a microorganism, such as an algae, an archaea-bacteria, a bacterium, a fungus, a protozoan, a virus. In one embodiment, the microorganism is a pathogen for plants, for animals, and in particular for humans.

[0035] In one embodiment, a bacterium pathogenic to an animal is selected from the non-exhaustive list comprising a bacterium of the species Afipiafelis; a a bacterium of the genus Anaplasma, preferably a bacterium of the species Anaplasma pha-gocytophilum, Anaplasma centrale, Anaplasma mesaeterum, Anaplasma platys, Anaplasma bovis, or Anaplasma ovis; 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 pseudomallei; 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 bumetii; 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 Erancisella, preferably a bacterium of the species Erancisella tularensis; a bacterium of the genus Haemophilus, preferably a bacterium of the species Haemophilus ducreyi or Haemophilus influenzae; a bacterium of the genus Helicobacter, preferably a bacterium of the species Helicobacter pylori; a bacterium of the genus Eegionella, preferably a bacterium of the species Eegionella pneumophila; a bacterium of the genus Leptospira, preferably a bacterium of the species Eeptospira interrogans; a bacterium of the genus Eisteria, preferably a bacterium of the species Eisteria 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 Mycoplasmapneumoniae 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 Pas-; teurella, 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 Shigelladysenteriae, Shigella flexneri, 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; 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 whipplei; 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 enterocolitica, or Yersinia pseudotuberculosis.

[0036] In one embodiment, a fungus pathogenic to an animal is selected from the non-exhaustive list comprising a fungus of the genus Aspergillus, a fungus of the genus Candida.

[0037] In one embodiment, a protozoan pathogenic to an animal is selected from the non-exhaustive list comprising a protozoan of the genus Babesia, a protozoan of the genus Plasmodium, a protozoan of the genus Theileria, a protozoan of the genus Toxoplasma.

[0038] In practice, the virus may be a DNA virus or an RNA virus.

[0039] In one embodiment, the DNA virus is selected from the non- exhaustive comprising a virus of the family Adenoviridae, preferably a virus of the genus Adenovirus; of the family Hepdnaviridae, preferably a virus of the genus Herpadnavirus; of the family Herpesviridae, preferably a virus of the genus Herpesvirus; of the family Papovaviridae, preferably a virus of the genus Poly-omavirus or Papillomavirus; of the family Parvoviridae, preferably a virus of the genus Parvovirus; of the family Poxviridae, preferably a virus of the genus Poxvirus.

[0040] In one embodiment, the RNA virus is selected from the non-exhaustive list comprising 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; of the Filoviridae family, preferably a virus of the Filovirus genus; of the Flaviviridae family, preferably a virus of the genus Flavivirus, Ribivirus or Hepcivirus ; of the family Orthomyxoviridae, preferably a virus of the genus Influenzavirus; of the family Paramyxoviridae, preferably a virus of the genus Pneumovirus, Paramyxovirus or Morbillivirus; of the family Picornaviridae, preferably a virus of the genus Enterovirus, Hepatovirus or Rhinovirus; of the family Rhabdoviridae, preferably a virus of the genus Lyssavirus or Vesiculovirus; of the family Reoviridae, preferably a virus of the genus Reovirus or Rotavirus; of the family Retroviridae, preferably a virus of the genus Oncomavirus or Lentivirus; of the family Togaviridae, preferably a virus of the genus Alphavirus.

[0041] In one embodiment, the virus is selected from the non-exhaustive list comprising African horse sickness virus, African swine fever virus, Andes virus, avian influenza virus, 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 virus), swine fever virus, Japanese encephalitis virus, Junin, 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, Monkeypox virus, Murray Valley encephalitis virus, Newcastle disease virus, Nipah virus, Omsk hemorrhagic fever virus, Oropouche virus, Peste des petits ruminants virus, Porcine enterovirus type 9 (or swine vesicular disease virus), Powassan encephalitis virus, Rabies virus, Rift Valley virus, Rinderpest virus, Rocio virus, Sabia virus, Seoul virus, Sheeppox virus, Sin Nombre virus, St. Louis encephalitis virus, Teschen disease virus, Tick-borne encephalitis virus, Smallpox virus, Equine encephalitis virus Venezuela, vesicular stomatitis virus, western equine encephalitis virus and yellow fever virus.

[0042] In practice, the sample can be of very varied origin.

[0043] In some embodiments, the sample is from an animal, preferably a mammal, preferably a human. In one embodiment the mammal is selected from the non-exhaustive list comprising a cat, a horse, a goat, a dog, a guinea pig, a rabbit, a sheep, a pig, a rat, a mouse, a cow, a poultry. In one embodiment the mammal is a pet animal selected from the non-exhaustive list comprising a cat, a dog, a guinea pig, a rat, a mouse. In one embodiment the mammal is an animal of economic interest, for example selected from the non-exhaustive list comprising a horse, a rabbit, a sheep, a pig, a cow, a poultry.

[0044] In one embodiment, the biological, environmental or agri-food samples are taken in compliance with good practices and possibly current regulatory practices.

[0045] For example, sterile conditions may be desired. Samples may be collected using a suitable instrument, including but not limited to, a syringe, toothpick, swab, spatula, receptacle, forceps, scalpel, adhesive paper.

[0046] In one embodiment, the blood samples are stored in suitable tubes, for example containing heparin or EDTA, so as to prevent the coagulation of blood cells.

[0047] In one embodiment, the sample is stored before implementing the methods according to the present invention at a temperature between -196°C and 50°C, preferably between -196°C and 25°C, between -80°C and 25°C, between -20°C and 25°C, between 4°C and 25°C. In one embodiment, the sample is stored at room temperature before implementing the methods according to the present invention.

[0048] In one embodiment, the sample is stored before implementing the methods according to the present invention for a period not exceeding 7 days, preferably for a period not exceeding 6 days, 5 days, 4 days, 3 days, 2 days, 24 hours, 12 hours, 6 hours, 2 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 2 minutes.

[0049] In practice, the lysis buffer comprises an SDS concentration of between approximately 1% and 25%. In the context of the invention, the term "between approximately 1% and 25%" includes approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24% and 25%.

[0050] In the context of the invention, the lysis buffer is more particularly intended to promote the lysis of biological entities, e.g., cells, viruses, containing the nucleic acids of interest.

[0051] In practice, the concentration of the buffer comprising a potassium salt is between approximately 10 mM and 500 mM. As used herein, the term "between about 10 mM and 500 mM" includes about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 nM, 120 mM, 140 mM, 160 mM, 180 mM, 200 mM, 220 mM, 240 mM, 260 mM, 280 mM, 300 mM, 320 mM, 340 mM, 360 mM, 380 mM, 400 mM, 420 mM, 440 mM, 460 mM, 480 mM and 500 mM.

[0052] The buffer comprising the potassium salt is intended to precipitate the SDS which is in compatible with nucleic acid analysis steps implemented in many methods, such as amplification, particularly PCR amplification.

[0053] In practice, the potassium salt can advantageously be chosen from potassium acetate (CH3CO2K), potassium chloride (KC1), potassium hydrogen carbonate (CHKO3). These salts can be prepared extratemporaneously or alternatively be acquired commercially.

[0054] In one embodiment, the potassium salt is potassium hydrogen carbonate (CHKO3).

[0055] In practice, the concentration of the buffer comprising a zinc salt is between about 0.5 M and 5.0 M. In the context of the invention, the term "between about 0.5 M and 5.0 M" includes about 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M, 1.2 M, 1.4 M, 1.6 M, 1.8 M, 2.0 M, 2.2 M, 2.4 M, 2.6 M, 2.8 M, 3.0 M, 3.2 M, 3.4 M, 3.6 M, 3.8 M, 4.0 M, 4.2 M, 4.4 M, 4.6 M, 4.8 M and 5.0 M.

[0056] In the context of the invention, the buffer comprising the zinc salt is particularly intended to precipitate the hemoglobin contained in blood samples or certain samples containing blood, such as for example biopsies. Indeed, hemoglobin is known to be incompatible with certain steps of nucleic acid analysis implemented in many methods, such as for example amplification, in particular amplification by PCR.

[0057] In practice, the potassium salt can advantageously be chosen from zinc carbonate (ZnCO3), zinc chloride (ZnCl), zinc sulfate (ZnSO4). These salts can be prepared extratemporaneously or alternatively be acquired commercially.

[0058] In one embodiment, the zinc salt is zinc sulfate (ZnSO4).

[0059] In practice, the filter having a pore diameter of between approximately 1 μm and 10 pm. As used herein, the term "between about 1 pm and 10 pm" includes about 1 pm, 2 pm, 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, and 10 pm.

[0060] In one embodiment, the filter has a membrane having pores with a diameter of between about 2 pm and 10 pm, in particular a diameter of about 5 pm.

[0061] The filter is more particularly intended to retain the precipitate which contains SDS, the potassium salt, and possibly the insoluble cellular debris. When the sample is a blood sample or a sample containing blood, the filter is also intended to retain the precipitate which contains hemoglobin and zinc. On the contrary, the filter is intended to allow the nucleic acids contained in the sample to pass through.

[0062] In some embodiments, the filter membrane is made of cellulose acetate, cellulose nitrate, nylon, polyamide, silica. Filters suitable for implementing the invention can be purchased commercially, for example from Sartorius®, Hahnemühle®, Sebio®.

[0063] In one embodiment, at least one of the buffers of the kit, i.e. the lysis buffer, the buffer containing a potassium salt, the buffer containing a zinc salt, comprises an RNase inhibitor. This embodiment is particularly advantageous when the nucleic acids of interest contained in the sample are ribonucleic acids (RNAs).

[0064] The invention also relates to the use of a kit according to the invention in a method for extracting nucleic acids contained in a complex sample.

[0065] The methods according to the invention are implemented in vitro or ex vivo.

[0066] The invention relates to a method for extracting nucleic acids contained in a complex sample, said method implementing the kit according to the invention.

[0067] The invention also relates to a method for extracting nucleic acids contained in a complex sample, said method comprising the following steps:

[0068] - a) contacting the complex sample with a lysis buffer comprising an SDS concentration of between approximately 1% and 25%, such that the final SDS concentration in the reaction mixture is between approximately 0.01% and 10.0%; - b) optionally contacting the mixture from step a) with a buffer comprising a concentration of a zinc salt of between approximately 0.5 M and 5.0 M, so that the final concentration of zinc salt in the reaction mixture is between approximately 10 mM and 70 mM; - c) contacting the mixture from step a) or b) with a buffer comprising a concentration of a potassium salt of between approximately 0.1 M and 5.0 M, so that the final concentration of potassium salt in the reaction mixture is between approximately 10 mM and 500 mM; - d) filtering the reaction mixture from step c) so as to collect soluble nucleic acids.

[0069] In the context of the invention, the complex sample in step a) comprises at least 25 copies of a nucleic acid, preferably at least 10 copies of a nucleic acid, preferably at least 1 copy of a nucleic acid to be extracted.

[0070] In one embodiment, the method is implemented using a kit according to the invention.

[0071] In the context of the invention, the term “reaction mixture” means the mixture of all the ingredients at the end of step c). Thus, the “reaction mixture” within the meaning of the invention comprises the sample containing the nucleic acids of interest, the lysis buffer, the buffer comprising the potassium salt and optionally the buffer including zinc salt.

[0072] In practice, the final SDS concentration in the reaction mixture is between approximately 0.01% and 10.0%. As used herein, the term "between about 0.1% and 10.0%" includes 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% and 10.0%.

[0073] In practice, the final concentration of zinc salt in the reaction mixture is between approximately 10 mM and 70 mM. In the context of the invention, the term "between approximately 10 mM and 70 mM" includes approximately 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM and 70 mM.

[0074] In practice, the final concentration of potassium salt in the reaction mixture is between approximately 10 mM and 500 mM. As used herein, the term "between about 10 mM and 500 mM" includes about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 140 mM, 160 mM, 180 mM, 200 mM, 220 mM, 240 mM, 260 mM, 280 mM, 300 mM, 320 mM, 340 mM, 360 mM, 380 mM, 400 mM, 420 mM, 440 mM, 460 mM, 480 mM and 500 mM.

[0075] In one embodiment, the complex sample is selected from the group comprising a biological, environmental, agri-food sample, preferably a biological sample.

[0076] In some embodiments, the sample is a sample of a biological fluid. In some embodiments, the biological fluid is selected from the group consisting of bile, feces, aqueous humor, milk, amniotic fluid, lymph, cerebrospinal fluid, plasma, respiratory lavage product, guttural pouch product, pus, nasopharyngeal secretion, lacrimal secretion, vaginal secretion, saliva, blood, serum, semen, and urine. In one embodiment, the biological fluid is selected from the group consisting of feces, saliva, nasopharyngeal secretion, blood.

[0077] In some embodiments, the biological fluid is blood.

[0078] In one embodiment, step b) is implemented when the complex sample is a blood sample.

[0079] In one embodiment, the final SDS concentration in the reaction mixture is between about 0.01% and 1%, preferably between about 0.25% and 0.75%, when the complex sample is a blood sample.

[0080] As used herein, the term "between about 0.01% and 1%" includes about 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%, 0.08%, 0.085%, 0.09%, 0.095%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95% and 1%.

[0081] In one embodiment, the final potassium salt concentration in the reaction mixture is between about 10 mM and 500 mM, preferably between about 25 mM and 150 mM, when the complex sample is a blood sample.

[0082] In some embodiments, the biological fluid is selected from the group consisting of feces, saliva, and nasopharyngeal secretion.

[0083] In one embodiment, the final SDS concentration in the reaction mixture is between about 0.06% and 10%, preferably between about 0.1% and 6.7%, when the complex sample is selected from the group consisting of a fecal sample, a saliva sample and a nasopharyngeal secretion sample.

[0084] As used herein, the term "between about 0.06% and 10%" includes about 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, 0.5%, 0.55%, 0.6%, 0.65%, 0.7%, 0.75%, 0.8%, 0.85%, 0.9%, 0.95%, 1%, 1.2%, 1.4%, 1.6%, 1.8%, 2%, 2.2%, 2.4%, 2.6%, 2.8%, 3%, 3.2%, 3.4%, 3.6%, 3.8%, 4%, 4.2%, 4.4%, 4.6%, 4.8%, 5%, 5.2%, 5.4%, 5.6%, 5.8%, 6%, 6.2%, 6.4%, 6.6%, 6.8%, 7%, 7.2%, 7.4%, 7.6%, 7.8%, 8%, 8.2%, 8.4%, 8.6%, 8.8%, 9%, 9.2%, 9.4%, 9.6%, 9.8% and 10%.

[0085] In one embodiment, the final potassium salt concentration in the reaction mixture is between about 60 mM and 300 mM, preferably between about 100 mM and 270 mM, when the complex sample is selected from the group consisting of a fecal sample, a saliva sample and a nasopharyngeal secretion sample.

[0086] As used herein, the term "between about 60 mM and 300 mM" includes about 60 mM, 80 mM, 100 mM, 120 mM, 140 mM, 160 mM, 180 mM, 200 mM, 220 mM, 240 mM, 260 mM, 280 mM and 300 mM.

[0087] In one embodiment, the mixture at the end of step a), optionally step b) and step c) is homogenized, preferably by gentle stirring, more preferably by inverting the tube.

[0088] In practice, the implementation of the method according to the invention makes it possible to obtain a solution of nucleic acids in less than approximately 5 min, preferably in less than approximately 3 min, preferably in less than approximately 2 min.

[0089] In practice, the method can be carried out at room temperature. In the context of the invention, the expression "room temperature" includes a temperature between approximately 5°C and 40°C, i.e. temperatures of approximately 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 12°C, 14°C, 16°C, 18°C, 20°C, 22°C, 24°C, 26°C, 28°C, 30°C, 32°C, 34°C, 36°C, 38°C and 40°C.

[0090] The kit and the method according to the invention can be implemented in various circumstances:

[0091] - Identify the presence of a potentially pathogenic microorganism in a biological, environmental or agri-food sample; - Carry out a metagenomic analysis, i.e. analyze the composition of living organisms in a sample; - Identify one or more biomarker(s) associated with a pathology in a biological sample.

[0092] In practice, the method according to the invention will be preferred when access to laboratory equipment is difficult, or when the urgent nature of the analysis of the nucleic acids of interest requires rapid action.

[0093] The nucleic acids extracted by a kit or method according to the invention can subsequently be analyzed to identify the presence of a potentially pathogenic microorganism and thus diagnose an infectious pathology.

[0094] In certain embodiments, the extraction of nucleic acids according to the invention can be implemented for the diagnosis of a canine pathology selected from the group comprising anaplasmosis (bacteria of the genus Anaplasma), canine coronarosis, eczema of parasitic origin, canine ehrlichiosis (bacteria of the species Ehrlichia canis), mange (mites: Otodecte cynotis, Sarcoptes scabiei, Dermodex canis).gastroenteritis, giardiasis (parasite of the genus Giardia), hepatitis (adenovirus CAV1), canine hemobartonellosis, leishmaniasis (Leshmania Infantum), leptospirosis (bacteria of the genus Eeptospira), distemper (virus of the genus Morbillivirus), Lyme disease or borreliosis (bacteria of the species Borrelia Burgdorferi), otitis, parvovirosis, piroplasmosis or babesiosis (parasite Babesia canis), rabies (rabies virus Lyssavirus), spirocercosis (parasite Spirocerca Lupi), ringworm (fungus Microsporum canis, Trichophyton mentagrophytes, Microsporum gypseum), tapeworm (worm Toenia, Dipylidium, Echinococcus), tetanus (bacteria Clostridium tetani). kennel cough (association of the canine Parainfluenza virus or an adenovirus with the bacterium Bordetella Bronchiseptica or Pseudomonaaeruginosa), and tuberculosis (bacteria Mycobacterium bovis or Mycobacterium tuberculosis).

[0095] In certain embodiments, the extraction of nucleic acids according to the invention can be implemented for the diagnosis of a feline pathology selected from the group comprising anaplasmosis, bordetellosis, borreliosis, calicivirosis, feline chlamydophilosis, conjunctivitis, chlamydiosis, coronavirosis, echinococcosis, encephalitis, giardiasis, gingivitis, herpes virus, hemobartonellosis, ear mange or otacariosis or ear otitis, feline leukemia (FeLV), piroplasmosis, feline rabies, feline viral rhinotracheitis (FVR), rickettsiosis, feline AIDS (FIV), feline urological syndrome (UFS), ringworm, toxoplasmosis, feline tritrichomoniasis, tuberculosis, and typhus.

[0096] In certain embodiments, the extraction of nucleic acids according to the invention can be implemented for the diagnosis of an equine pathology selected from the group comprising anaplasmosis, babesiosis, ehrlichiosis, equine influenza, strangles, leptospirosis, Lyme disease, piroplasmosis, rhinopneumonia, theileriosis.

[0097] The nucleic acids extracted using the kit or method according to the invention can be analyzed by any suitable technique described in the state of the art, or a derived technique. By way of illustration, a suitable technique for analyzing a nucleic acid can implement an amplification reaction, such as a thermal amplification reaction. Examples of thermal amplification reactions include, but are not limited to: the polymerase chain reaction (or 'Polymerase Chain Reaction', PCR) and its variants, eg, Hot-start PCR, Touchdown PCR, Nested PCR, Fast PCR, Direct PCR, Multiplex PCR, Long PCR, GC-rich PCR, Inverse PCR, Quantitative PCR, Ligation-Mediated PCR, Multiplex ligation-dependent probe amplification PCR, Methylation-specific PCR, Anchored PCR, Asymmetric PCR, Thermal Asymmetric Interlaced PCR, Assembly PCR, Allel-Specified PCR, Arbitrarily Primed PCR.

[0098] In practice, the amplification reaction may be an isothermal amplification reaction. Examples of isothermal amplification reactions include, but are not limited to: Loop Mediated Isothermal Amplification (LAMP), Recombinase Polymerase Amplification (RPA), Nucleic Acid Sequence-Based Amplification (NASBA), Helicase-Dependent Amplification (HDA), Rolling Circle Amplification (RCA), Strand Displacement Amplification (SDA) and Multiple Displacement Amplification (MDA).

[0099] In practice, the amplification reaction is an isothermal amplification reaction of the loop-mediated isothermal amplification (LAMP) type, in particular as described by Notomi et al. (Nucleic Acids Res., 2000, 28(12):e63, by patent EP1020534, or by Nagamine et al. (Mol Cell Probes., 2002, 16(3):223-9).

[0100] The LAMP amplification reaction uses a strand displacement polymerase, typically a Bst polymerase (extracted from the bacterium Bacillus stearothermophilus), Aac, phi-29 Vent, or a variant thereof. The LAMP amplification reaction can also be performed directly for amplification of RNA to DNA by adding a reverse transcriptase to the reaction mixture. An example of a reverse transcriptase includes, but is not limited to, AMV reverse transcriptase. In one embodiment, the polymerase used has both DNA- and RNA-dependent DNA polymerase activity, and the amplification reaction does not require the addition of a reverse transcriptase.

[0101] Kits allowing the implementation of the LAMP amplification reaction are commercially available and accessible to those skilled in the art.

[0102] For illustration purposes, the LAMP amplification reaction is carried out at a constant temperature of 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 higher.

[0103] By way of illustration, the LAMP amplification reaction is carried out for a duration of at least 2 minutes, at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes or more. In one embodiment, the LAMP amplification reaction is carried out for a duration of between 10 minutes and 60 minutes, preferably between 15 minutes and 45 minutes.

[0104] By way of illustration, the LAMP amplification reaction is carried out using an isothermal amplification system. Several instruments of this type are available on the market and accessible to those skilled in the art.

[0105] By way of illustration, the amplification reaction of the nucleic acids extracted according to the methods of the present invention comprises a step of detecting the amplicon, i.e., a step of detecting the product of the amplification of the target nucleotide sequence.

[0106] Techniques for detecting an amplicon are well known to those skilled in the art, and include, but are not limited to: the use of DNA intercalating agents (eg, ethidium bromide, propidium iodide, SYBR green, SYBR gold, crystal violet, GelRed, DAPI), the measurement of colorimetry and / or fluorescence, the use of oligonucleotide probes labeled with fluorescence, the presence of an antigen or radioactivity, the use of dNTPs labeled with fluorescence or radioactivity, enzymatic tests, analysis of the variation of absorbance, turbidimetry and lateral flow tests.

[0107] In practice, the amplicon detection step involves the use of DNA intercalating agents. According to this embodiment, the detection of the amplicon can be carried out with the naked eye, by observing the colorimetry of the sample, or by measuring the fluorescence emitted by the intercalating agent. For example, the addition of an intercalating agent SYBR green type intercalator colors the sample orange in the absence of intercalation (i.e., in the absence of amplification of a target sequence) or green / blue in the presence of intercalation (i.e., in the presence of amplification of a target sequence) (see for example, Iwamoto et al.; J Clin Microbiol., 2003, 41(6):2616-22). This type of intercalating agent can also be detected by fluorescence, which is emitted only when the agent intercalates between the nucleotide bases of a double strand of DNA (i.e., in the presence of amplification of a target sequence).

[0108] Alternatively, the amplicon detection step implements the use of fluorescence-labeled oligonucleotide probes and comprises the detection and / or measurement of fluorescence. According to this embodiment, the oligonucleotide probes are capable of hybridizing to the amplicon. BRIEF DESCRIPTION OF THE FIGURES

[0109] [fig.l][fig.l] is a diagram showing the protocol for extracting nucleic acids from a complex sample, according to the invention. From left to right, all or part of a sample is brought into contact with a lysis buffer, containing SDS, thus forming a first reaction mixture (step 1). For blood samples or samples containing blood, the presence of Zinc, in the first reaction mixture, makes it possible to precipitate hemoglobin. The SDS contained in the first reaction mixture is then precipitated using a buffer containing potassium, which is introduced into the first reaction mixture (step 2). The second reaction mixture at the end of the step of precipitation of SDS by potassium is then filtered using a suitable filter (step 3). The SDS / potassium precipitate is retained by the filter, as is the hemoglobin / Zinc precipitate, when the sample is a blood sample or containing blood.The nucleic acids contained in the second reaction mixture and derived from the sample are not retained by the filter and can be collected in a suitable receptacle, for example a tube. The nucleic acids extracted from the sample can be subjected to further analysis (step 4). EXAMPLES .

[0110] The present invention will be better understood by reading the following examples which illustrate the invention in a non-limiting manner. [YES] Example 1: Extraction of nucleic acids from a cat fecal sample 1.1 Materials and Methods

[0112] A sample of cat feces is collected with a sterile toothpick. The fecal matter on the toothpick is introduced into a tube containing 2 mL of a lysis buffer containing SDS. The toothpick is shaken for a few seconds to move the fecal matter from the toothpick to the lysis buffer. The toothpick is then removed. The tube containing the reaction mixture is then shaken to homogenize the fecal matter in the buffer. 1 mL of a buffer containing CHKO3 is then added to the reaction mixture, which is gently shaken by inverting the tube 3 or 4 times. The reaction mixture has a final volume of 3 mL. The contents of the tube are then poured into a 5 mL syringe at the end of which is a cellulose acetate filter with a pore diameter of approximately 5 μm (Sartorius®). Filtration is carried out so that the filtrate is collected in a sterile tube. Nucleic acids are analyzed as follows. The presence of the feline housekeeping gene encoding NADH dehydrogenase subunit 5 (ND5S) (GenBank accession number: NC_001700.1) is determined after isothermal amplification at 65 °C for 30 min. 1.2 Results

[0113] Table 1: Determination of effective SDS and CHKO3 concentrations for the extraction of nucleic acids from a cat feces sample

[0114] [Tables 1] Conc. SDS (%)1 Amplification Conc. CHKO 3 (mM)1 Amplification 0.067% + (2 / 2) 67 mM + (2 / 2) 0.67% +(3 / 3) 133 mM + (5 / 5) 1.3% +(3 / 3) 267 mM + (2 / 5) 3.33% + (2 / 2) 533 mM -(1 / 1) 6.67% + (1 / 2) 13.3% - (2 / 2)

[0115] 'Final concentration in the final reaction medium comprising feces, the buffer comprising SDS and the buffer comprising CHKO3 (total final volume of the reaction medium before filtration 3 mL).

[0116] In conclusion, the final concentration of SDS in the reaction medium is between approximately 0.06% and 10%, and that of CHKO3 is between approximately 60 mM and 300 mM, for an extraction of nucleic acids from a sample of animal feces.

[0117] Example 2: Determination of the effective SDS concentration to ensure the extraction of nucleic acids from a blood sample

[0118] Freshly collected blood samples are immediately brought into contact with a buffer comprising 20% ​​SDS. The final SDS concentrations in the reaction mixture are between 0.33% and 2.5%, i.e. 0.33%, 1.67%, 2.5%. The A final SDS concentration of 0.33% produces a clear lysate. In contrast, final SDS concentrations of 1.67% and 2.5% lead to blood cell coagulation.

[0119] In conclusion, the final SDS concentration in the reaction medium must not be greater than 1%, for an extraction of nucleic acids from an animal blood sample.

[0120] Example 3: Determination of the effective concentrations of ZnSO ^ and CHKO 3 for the extraction of nucleic acids from a horse blood sample 3.1 Materials and Methods

[0121] A horse blood sample is collected according to the usual protocol. To 0.6 mL of collected blood are added 30 pL of a lysis buffer containing 20% ​​SDS and a volume of 30 pL of a buffer containing ZnSO4. The tube containing the reaction mixture is then gently shaken by inverting the tube 3 or 4 times, so as to homogenize it. 1.14 mL of a buffer containing CHKO3 is then added to the reaction mixture, which is gently shaken by inverting the tube 3 or 4 times. When the ZnSO4 concentration varies, the final CHKO3 concentration is fixed at 100 mM. When the CHKO3 concentration varies, the final ZnSO4 concentration is fixed at 33 mM. The reaction mixture (final volume 1.8 mL) is filtered as described in Example 1. The presence of the equine housekeeping gene encoding microglobulin [32 (B2M gene) (GenBank accession number: AH011712.2) is determined after isothermal amplification at 65 °C for 30 min. 3.2 Results

[0122] Table 2: Determination of effective ZnSO4 and CHKO3 concentrations for the extraction of nucleic acids from a horse blood sample

[0123] [Tables 2] Conc. ZnSO 4 (mM)1 Amplification Conc. CHKO 3 (mM)1 Amplification 8 mM + (3 / 4) OmM -(1 / 1) 10 mM + (1 / 1) 8 mM + (1 / 1) 17 mM + (1 / 1) 17 mM + (1 / 1) 25 mM + (1 / 2) 25 mM + (2 / 2) 30 mM + (1 / 4) 32 mM + (1 / 1) 32 mM + (2 / 2) 37 mM + (1 / 1) 33 mM + (11 / 12) 40 mM + (1 / 1) 40 mM - (2 / 2) 47.5 mM + (1 / 1) 50 mM - (3 / 3) 50 mM + (1 / 1) 70 mM - (3 / 3) 100 mM + (11 / 12) 116 mM + (6 / 10) 133 mM + (1 / 2)

[0124] 'Final concentration in the final reaction medium comprising blood, buffer comprising SDS, buffer comprising ZnSO4 and buffer comprising CHKO3 (total final volume before filtration 1.8 mL).

[0125] In conclusion, the final concentration of ZnSO4 in the reaction medium is between approximately 8 mM and 40 mM, and that of CHKO3 is greater than 8 mM, for an extraction of nucleic acids from a horse blood sample.

[0126] Example 4: Extraction of nucleic acids from a cat blood sample

[0127] A sample of cat blood is taken according to veterinary practice. To 1 mL of blood collected are added 50 pL of a lysis buffer containing 20% ​​SDS and 50 pL of a buffer containing ZnSO4. The tube containing the reaction mixture is then gently shaken by inverting the tube 3 or 4 times, so as to homogenize it. 2 mL of a buffer containing CHKO3 are then added to the reaction mixture, which is gently shaken by inverting the tube 3 or 4 times. The reaction mixture is then filtered as in Example 1 or 3, and the presence of the feline domestic gene encoding ND5S is demonstrated after isothermal amplification at 65 °C for 30 min.

[0128] Example 5: Extraction of nucleic acids from a dog blood sample 5.1 Materials and Methods

[0129] A dog blood sample is collected according to veterinary practice and processed as described in Example 4. When the ZnSO4 concentration varies, the final CHKO3 concentration is set at 40 mM. When the CHKO3 concentration varies, the final ZnSO4 concentration is set at 33 mM. The presence of the canine housekeeping gene encoding the NADH mbiquinone oxidoreductase core subunit 5 (GenBank accession number: AAU12157.1) is determined after isothermal amplification at 65 °C for 30 min. 5.2 Results

[0130] Table 3: Determination of effective ZnSO4 and CHKO3 concentrations for the extraction of nucleic acids from a dog blood sample

[0131] [Tables3] Conc. ZnSO 4 (mM)1 Amplification Conc. CHKO 3 (mM)1 Amplification 25 mM + (1 / 1) 25 mM + (1 / 1) 33 mM + (51 / 54) 33 mM + (1 / 1) 42 mM -(1 / 1) 40 mM + (51 / 54) 43 mM -(1 / 1) 50 mM -(1 / 1) 59 mM -(1 / 1) 67 mM -(1 / 1)

[0132] 'Final concentration in the final reaction medium comprising blood, buffer comprising SDS, buffer comprising ZnSO4 and buffer comprising CHKO3 (total final volume before filtration 3 mL).

[0133] In conclusion, the final concentration of ZnSO4 in the reaction medium must be less than 42 mM, and that of CHKO3 can be between approximately 25 mM and 40 mM, for an extraction of nucleic acids from a dog blood sample.

Claims

Claims

1. A kit for extracting nucleic acids contained in a complex sample, said kit comprising: - a lysis buffer comprising a concentration of SDS of between about 1% and 25%, preferably between about 5% and 20%; - a buffer comprising a concentration of a potassium salt of between about 0.1 M and 5.0 M; - a buffer comprising a concentration of a zinc salt of between about 0.5 M and 5.0 M; - a filter having a pore diameter of between about 1 pm and 10 pm, preferably between about 2 pm and 7 pm; - optionally, an element selected from a syringe for use with the filter, one or more reaction tubes, an instruction guide, and a combination thereof.

2. A kit according to claim 1, wherein the potassium salt is potassium hydrogen carbonate (CHKO3) and / or the zinc salt is zinc sulfate (ZnSO4).

3. Use of a kit according to one of claims 1 or 2, in a method for extracting nucleic acids contained in a complex sample.

4. A method for extracting nucleic acids contained in a complex sample, said method comprising the following steps: - a) contacting the complex sample with a lysis buffer comprising a concentration of SDS of between about 1% and 25%, such that the final concentration of SDS in the reaction mixture is between about 0.01% and 10%; - b) optionally contacting the mixture of step a) with a buffer comprising a concentration of a zinc salt of between about 0.5 M and 5.0 M, such that the final concentration of zinc salt in the reaction mixture is between about 10 mM and 70 mM; - c) contacting the mixture of step a) or b) with a buffer comprising a concentration of a potassium salt of between about 0.1 M and 5.0 M, such that the final concentration of potassium salt in the reaction mixture is between about 25 mM and 300 mM, preferably between about 25 mM and 150 mM or between about 60 mM and 300 mM; - d) filtering the reaction mixture of step c) so as to collect soluble nucleic acids.

5. Method according to claim 4, wherein the complex sample is selected from the group comprising a biological, environmental, agri-food sample, preferably a biological sample.

6. A method according to claim 4 or 5, wherein step b) is carried out when the complex sample is a blood sample.

7. A method according to any one of claims 4 to 6, wherein the final concentration of SDS in the reaction mixture is between about 0.01% and 1%, preferably between about 0.25% and 0.75%, when the complex sample is a blood sample.

8. A method according to any one of claims 4 to 7, wherein the final concentration of potassium salt in the reaction mixture is between about 25 mM and 150 mM, when the complex sample is a blood sample.

9. A method according to any one of claims 4 and 5, wherein the final SDS concentration in the reaction mixture is between about 0.06% and 10%, preferably between about 0.1% and 6.7%, when the complex sample is selected from the group consisting of a fecal sample, a saliva sample and a nasopharyngeal secretion sample.

10. A method according to any one of claims 4, 5 and 9, wherein the final concentration of potassium salt in the reaction mixture is between about 60 mM and 300 mM, preferably between about 100 mM and 270 mM, when the complex sample is selected from the group consisting of a fecal sample, a saliva sample and a nasopharyngeal secretion sample.

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