KITS AND METHODS FOR EXTRACTING NUCLEIC ACIDS FROM COMPLEX SAMPLES KITS AND METHODS FOR EXTRACTING NUCLEIC ACIDS FROM COMPLEX SAMPLES

Abstract

A kit and method for extracting nucleic acids from complex samples. The kit includes: (i) a lysis buffer having a concentration of SDS ranging from about 1% to about 25%; (ii) a buffer having a concentration of a potassium salt of ranging from about 0.1 M to about 5.0 M; (iii) a buffer having a concentration of a zinc and/or copper salt of ranging from about 0.5 M to about 5.0 M; (iv) a filter having a pore diameter ranging from about 1 μm to about 10 μm; and optionally, a member selected from one or more syringe(s), one or more reaction tube(s), an instruction guide, and any combination thereof.

Claims

1.-15. (canceled)

16. A kit for extracting nucleic acids contained in a complex sample, said kit comprising: a lysis buffer comprising a concentration of SDS ranging from about 1% to about 25%, preferably from about 2.5% to about 20%; a buffer comprising a concentration of a potassium salt ranging from about 0.1 M to about 5.0 M; a buffer comprising a concentration of a zinc and/or copper salt ranging from about 0.5 M to about 5.0 M; a filter having a pore diameter ranging from about 1 μm to about 10 μm, preferably from about 2 μm to about 7 μm; optionally, an item chosen from the group comprising or consisting of one more syringe(s), one or more reaction tube(s), an instruction guide, and any combination thereof.

17. The kit according to claim 16, wherein the zinc salt is selected from a group comprising or consisting of zinc sulfate (ZnSO.sub.4), and zinc chloride (ZnCl.sub.2).

18. The kit according to claim 16, wherein the copper salt is selected from a group comprising or consisting of copper sulfate (CuSO.sub.4), and copper chloride (CuCl.sub.2).

19. The kit according to claim 16, wherein the potassium salt is selected from the group comprising or consisting of potassium hydrogen carbonate (KHCO.sub.3), potassium acetate (CH.sub.3CO.sub.2K), dipotassium hydrogen phosphate (K.sub.2HPO.sub.4), monobasic potassium phosphate (KH.sub.2PO.sub.4), and potassium chloride (KCl).

20. The kit according to claim 16, wherein the lysis buffer and/or the buffer comprising a zinc and/or copper salt comprise(s) one or more additional compound(s) selected in the group comprising or consisting of Tris-HCl, HEPES, and MOPS.

21. A method for extracting nucleic acids contained in a complex sample, comprising extracting the nucleic acid using the kit according to claim 16.

22. 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 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%; b) optionally contacting the mixture from step a) 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 zinc and/or copper in the reaction mixture is ranging from about 10 mM to about 70 mM; c) contacting the mixture of step a) or b) 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 potassium salt in the reaction mixture is ranging from about 10 mM to about 500 mM; d) filtering the reaction mixture from step c); and e) collecting soluble nucleic acids.

23. The method according to claim 22, wherein the complex sample is selected from the group comprising a biological sample, environmental sample, food sample, preferably a biological sample.

24. The method according to claim 23, wherein the biological sample is blood.

25. The method according to claim 24, wherein step b) is performed when the complex sample is a blood sample.

26. The method according to claim 24, wherein the final concentration of SDS in the reaction mixture is ranging from about 0.01% to about 2%, preferably from about 0.01% to about 1.5%, preferably from about 0.01% to about 1%, preferably from about 0.25% to about 0.75%, when the complex sample is a blood sample.

27. The method according to claim 24, wherein the final concentration of potassium salt in the reaction mixture is ranging from about 10 mM to about 500 mM, preferably from about 25 mM to about 150 mM, when the complex sample is a blood sample.

28. The method according to claim 23, wherein the biological sample is selected from the group consisting of feces sample, saliva sample, a product of respiratory lavage sample, and a nasopharyngeal secretion sample.

29. The method according to claim 28, wherein 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 complex sample is selected from group consisting of a feces sample, a saliva sample, a respiratory lavage sample, and a nasopharyngeal secretion sample.

30. The method according to claim 28, wherein 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 complex sample is selected from the group consisting of a feces sample, a saliva sample, respiratory lavage sample, and a nasopharyngeal secretion sample.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0190] FIG. 1 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 contacted with a lysis buffer containing SDS, thus forming a first reaction mixture (step 1). For blood samples or samples containing blood, the addition of zinc and/or copper, in the first reaction mixture, allows hemoglobin to be precipitated. The SDS contained in the first reaction mixture is then precipitated by means of a buffer containing a potassium salt, which is introduced into the first reaction mixture (step 2). The second reaction mixture following the step of precipitating SDS with potassium is then filtered using a suitable filter (step 3). The SDS/potassium precipitate is retained by the filter, as is the hemoglobin/zinc and/or copper precipitate, when the sample is a blood sample or contains blood. The nucleic acids contained in the second reaction mixture and from the sample are not retained by the filter and can be collected in a suitable receptacle, for example a tube. Nucleic acids extracted from the sample can be analyzed in a later step (step 4).

EXAMPLES

[0191] The present invention is further illustrated by the following non-limitative examples and by FIG. 1.

Example 1: Extraction of Nucleic Acids from a Sample of Cat Feces

1.1 Materials and Methods

[0192] A sample of cat feces is collected with a sterile toothpick. The fecal matter contained on the toothpick is placed in a tube containing 2 mL of lysis buffer containing SDS. The toothpick is shaken for a few seconds so as 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, so as to homogenize the fecal matter with the buffer. 1 mL of a buffer containing KHCO.sub.3 is then added to the reaction mixture, which is gently stirred 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 domestic gene encoding the subunit 5 of NADH dehydrogenase (NDSS) (GenBank accession number: NC_001700.1) is determined after isothermal amplification at 65° C. for 30 min.

1.2 Results

[0193]

TABLE-US-00001 TABLE 1 Determination of the effective concentrations of SDS and KHCO.sub.3 for the extraction of nucleic acids from a sample of cat feces. SDS conc. (%).sup.1 Amplification.sup.2 KHCO.sub.3 conc. (mM).sup.1 Amplification.sup.2 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) .sup.1Final concentration in the final reaction mixture comprising the feces, the buffer comprising the SDS and the buffer comprising the KHCO.sub.3 (total final volume of the reaction mixture before filtration: 3 mL). .sup.2Number of assays in which specific amplification of the domestic gene is observed over the total number of assays.

[0194] In conclusion, the final concentration of SDS in the reaction mixture is between approximately 0.06% and 10%, and that of KHCO.sub.3 is between approximately 60 mM and 300 mM, for an extraction of nucleic acids from an animal's feces sample.

Example 2: Determination of the Effective SDS Concentration to Ensure the Extraction of Nucleic Acids from a Blood Sample

[0195] The freshly drawn blood samples are immediately contacted 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 final SDS concentration of 0.33% results in a clear lysate. In contrast, the final SDS concentrations of 1.67% and 2.5% lead to clotting of blood cells.

[0196] In conclusion, the final concentration of SDS in the reaction mixture should not be greater than 1%, for extraction of nucleic acids from an animal blood sample.

Example 3: Determination of the Effective Concentrations of ZnSO.SUB.4 .and KHCO.SUB.3 .for the Extraction of Nucleic Acids from a Sample of Horse Blood

3.1 Materials and Methods

[0197] A horse blood sample is collected according to the usual practices. To 0.6 mL of collected blood are added 30 μL of a lysis buffer containing 20% SDS and a volume of 30 μL of a buffer containing ZnSO.sub.4. The tube containing the reaction mixture is then gently stirred by inverting the tube 3 or 4 times, so as to homogenize. 1.14 mL of a buffer containing KHCO.sub.3 are then added to the reaction mixture, which is gently stirred by inverting the tube 3 or 4 times. When the ZnSO.sub.4 concentration varies, the final KHCO.sub.3 concentration is set at 100 mM. When the KHCO.sub.3 concentration varies, the final ZnSO.sub.4 concentration is set at 33 mM. The reaction mixture (final volume 1.8 mL) is filtered as described in Example 1. The presence of the equine domestic gene encoding (32 microglobulin (B2M gene) (GenBank accession number: AH011712.2) is determined after an isothermal amplification at 65° C. for 30 min.

3.2 Results

[0198]

TABLE-US-00002 TABLE 2 Determination of the effective ZnSO.sub.4 and KHCO.sub.3 concentrations for the extraction of nucleic acids from a horse blood sample ZnSO.sub.4 conc. (mM).sup.1 Amplification.sup.2 KHCO.sub.3 conc. (mM).sup.1 Amplification.sup.2  8 mM +(3/4)  0 mM −(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) .sup.1Final concentration in the final reaction mixture comprising blood, the buffer comprising SDS, the buffer comprising ZnSO.sub.4 and the buffer comprising KHCO.sub.3 (total final volume before filtration: 1.8 mL). .sup.2Number of assays in which specific amplification of the domestic gene is observed over the total number of assays.

[0199] In conclusion, the final concentration of ZnSO.sub.4 in the reaction mixture is between about 8 mM and 40 mM, and that of KHCO.sub.3 is greater than 8 mM, for extraction of nucleic acids from a horse blood sample.

Example 4: Extraction of Nucleic Acids from a Cat Blood Sample

[0200] A cat blood sample is collected according to veterinary practice. To 1 mL of collected blood are added 50 μL of a lysis buffer containing 20% SDS and 50 μL of a buffer containing ZnSO.sub.4. The tube containing the reaction mixture is then gently stirred by inverting the tube 3 or 4 times, so as to homogenize. 2 mL of a buffer containing KHCO.sub.3 are then added to the reaction mixture, which is gently stirred 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 shown after isothermal amplification at 65° C. for 30 min.

Example 5: Extraction of Nucleic Acids from a Dog Blood Sample

5.1 Materials and Methods

[0201] A dog blood sample is collected according to veterinary practice, and treated as described in Example 4. When the ZnSO.sub.4 concentration varies, the final KHCO.sub.3 concentration is set at 40 mM. When the KHCO.sub.3 concentration varies, the final ZnSO.sub.4 concentration is set at 33 mM. The presence of the canine domestic gene encoding the NADH core subunit 5: ubiquinone oxidoreductase (GenBank accession number: AAU12157.1) is determined after isothermal amplification at 65° C. for 30 min.

5.2 Results

[0202]

TABLE-US-00003 TABLE 3 Determination of the effective concentrations of ZnSO.sub.4 and KHCO.sub.3 for the extraction of nucleic acids from a dog blood sample ZnSO.sub.4 conc. (mM).sup.1 Amplification.sup.2 KHCO.sub.3 conc. (mM).sup.1 Amplification.sup.2 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) .sup.1Final concentration in the final reaction mixture comprising blood, the buffer comprising SDS, the buffer comprising ZnSO.sub.4 and the buffer comprising KHCO.sub.3 (total final volume before filtration 3 mL). .sup.2Number of assays in which specific amplification of the domestic gene is observed over the total number of assays.

[0203] In conclusion, the final concentration of ZnSO.sub.4 in the reaction mixture should be less than 42 mM, and that of KHCO.sub.3 can be between about 25 mM and 40 mM, for extraction of nucleic acids from a blood sample of dog.

Example 6: Extraction of Nucleic Acids from Various Samples

6.1 Materials and Methods

[0204] Blood and feces samples are taken according to the examples above.

[0205] Swabs and samples originating from nasopharyngeal and throat pouch lavages were collected according to standard medical or veterinary practice. Samples from environment were collected by rubbing a swab against a table (on a surface equivalent of a 25 cm.sup.2 square) to collect all the particles available on this surface.

[0206] The presence of the feline domestic gene encoding the subunit 5 of NADH dehydrogenase (NDSS) (GenBank accession number: NC_001700.1); of the canine domestic gene encoding the NADH core subunit 5: ubiquinone oxidoreductase (GenBank accession number: AAU12157.1); of the equine domestic gene encoding (32 microglobulin (B2M gene) (GenBank accession number: AH011712.2) is determined after an isothermal amplification at 65° C. for 30 min.

[0207] The presence of the human domestic gene encoding the Homo sapiens ribonuclease P protein subunit p20 (RNAseP) (GenBank accession number: U94316.1) is determined after an isothermal amplification at 65° C. for 25 min.

[0208] Finally, the presence of a synthetic DNA sequence which does not exist in living organisms on earth was identified as a positive control. This synthetic DNA sequence was called “Alien”. The Alien sequence was synthetized and a small quantity of this DNA was added on the environment to be collected by the swab. The presence of a synthetic Alien DNA sequence in the environmental sample is determined after an isothermal amplification at 65° C. for 20 min

6.2 Results

[0209] The extraction conditions described hereunder in Table 4 and Table 5 all allowed significant amplification of the above-mentioned genes. This indicates that the extraction of nucleic acids from the complex samples was efficient.

TABLE-US-00004 TABLE 4 nucleic acid extraction from non-blood samples Potassium buffer Lysis buffer initial/final initial/final Sample’s origin concentration concentration Oral/feces swab (cat) or SDS: 5.0%/2.5% KHCO.sub.3: 0.4M/0.2M Nasopharyngeal swab (human) Oral/feces swab (cat) or SDS: 2.5%/1.22% KHCO.sub.3: 0.3M/0.146M Nasopharyngeal swab Tris-HCl 1M, pH (human) 7.5/0.025M Throat Pouch lavage SDS: 20.0%/2.4% KHCO.sub.3: 0.40M/0.21M (horse) Throat Pouch lavage SDS: 20.0%/2.4% KHCO.sub.3: 0.30M/0.159M (horse) Tris-HCl 1M, pH 7.5/0.023M Environmental swab SDS: 5.0%/2.5% KHCO.sub.3: 0.4M/0.2M

TABLE-US-00005 TABLE 5 nucleic acid extraction from blood samples Copper buffer Potassium buffer Sample’s Lysis buffer initial/final initial/final initial/final origin concentration concentration concentration Blood (cat/dog) SDS: 5.0%/1.25% CuSO.sub.4 KHCO.sub.3: Tris-HCl 1M, pH 1M/0.0375M 0.3M/0.15M 7.5/0.025M Blood (horse) SDS: 5%/1.25% CuSO.sub.4 KHCO.sub.3: Tris-HCl 1M, pH 1M/0.025M 0.3M/0.15M 7.5/0.025M

[0210] As a conclusion, the initial concentrations of SDS, and KHCO.sub.3 within their respective buffers may vary without affecting the nucleic acids' extraction yield. In addition, the presence of Tris-HCl was shown to improve the yield of extracted nucleic acids.

[0211] Finally, KHCO.sub.3 could be replaced with K.sub.2HPO.sub.4 or KH.sub.2PO.sub.4 without affecting the extraction (see Table 6 below).

TABLE-US-00006 TABLE 6 nucleic acid extraction from non-blood samples with various potassium salts Potassium buffer Lysis buffer initial/final initial/final Sample’s origin concentration concentration Oral swab (cat) or SDS: 2.5%/1.22% KHCO.sub.3: Nasopharyngeal swab Tris-HCl 1M, pH 0.3M/0.146M (human) 7.5/0.025M Oral swab (cat) or SDS: 2.5%/1.22% KH.sub.2PO.sub.4: Nasopharyngeal swab Tris-HCl 1M, pH 0.3M/0.146M (human) 7.5/0.025M Oral swab (cat) or SDS: 2.5%/1.22% K.sub.2HPO.sub.4: Nasopharyngeal swab Tris-HCl 1M, pH 0.15M/0.073M (human) 7.5/0.025M