CN116590280A - Candida nucleic acid extraction lysate, kit and extraction method - Google Patents
Candida nucleic acid extraction lysate, kit and extraction method Download PDFInfo
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- CN116590280A CN116590280A CN202310658440.5A CN202310658440A CN116590280A CN 116590280 A CN116590280 A CN 116590280A CN 202310658440 A CN202310658440 A CN 202310658440A CN 116590280 A CN116590280 A CN 116590280A
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- 239000006166 lysate Substances 0.000 title claims abstract description 104
- 241000222120 Candida <Saccharomycetales> Species 0.000 title claims abstract description 76
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 56
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 56
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 56
- 238000000605 extraction Methods 0.000 title claims abstract description 53
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical compound COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 claims abstract description 52
- 239000007788 liquid Substances 0.000 claims abstract description 37
- 238000000197 pyrolysis Methods 0.000 claims abstract description 29
- INHCSSUBVCNVSK-UHFFFAOYSA-L lithium sulfate Inorganic materials [Li+].[Li+].[O-]S([O-])(=O)=O INHCSSUBVCNVSK-UHFFFAOYSA-L 0.000 claims abstract description 26
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 claims abstract description 26
- 239000004292 methyl p-hydroxybenzoate Substances 0.000 claims abstract description 26
- 229960002216 methylparaben Drugs 0.000 claims abstract description 26
- RBTVSNLYYIMMKS-UHFFFAOYSA-N tert-butyl 3-aminoazetidine-1-carboxylate;hydrochloride Chemical compound Cl.CC(C)(C)OC(=O)N1CC(N)C1 RBTVSNLYYIMMKS-UHFFFAOYSA-N 0.000 claims abstract description 26
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 22
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 claims abstract description 16
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims abstract description 16
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 239000011780 sodium chloride Substances 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims description 65
- 238000002156 mixing Methods 0.000 claims description 28
- 239000000203 mixture Substances 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 19
- 239000012153 distilled water Substances 0.000 claims description 18
- 238000003756 stirring Methods 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 14
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 11
- 230000009089 cytolysis Effects 0.000 claims description 10
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 7
- 239000002244 precipitate Substances 0.000 claims description 7
- 238000007789 sealing Methods 0.000 claims description 7
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- 239000007983 Tris buffer Substances 0.000 claims description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 238000003776 cleavage reaction Methods 0.000 claims description 3
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- 238000005336 cracking Methods 0.000 abstract description 15
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- 238000001556 precipitation Methods 0.000 abstract description 3
- HBBGRARXTFLTSG-UHFFFAOYSA-N Lithium ion Chemical compound [Li+] HBBGRARXTFLTSG-UHFFFAOYSA-N 0.000 abstract description 2
- 230000008859 change Effects 0.000 abstract description 2
- 229910001416 lithium ion Inorganic materials 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 42
- 241000222122 Candida albicans Species 0.000 description 10
- 241000235645 Pichia kudriavzevii Species 0.000 description 10
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- 238000000246 agarose gel electrophoresis Methods 0.000 description 9
- 229940071106 ethylenediaminetetraacetate Drugs 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 7
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- 238000010257 thawing Methods 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
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- 108020004463 18S ribosomal RNA Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
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- QPJVMBTYPHYUOC-UHFFFAOYSA-N methyl benzoate Chemical compound COC(=O)C1=CC=CC=C1 QPJVMBTYPHYUOC-UHFFFAOYSA-N 0.000 description 2
- 210000004400 mucous membrane Anatomy 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
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- 238000012163 sequencing technique Methods 0.000 description 2
- IKHKJYWPWWBSFZ-UHFFFAOYSA-N 4-[[4-(diethylamino)phenyl]-(4-diethylazaniumylidenecyclohexa-2,5-dien-1-ylidene)methyl]benzene-1,3-disulfonate;hydron Chemical compound C1=CC(N(CC)CC)=CC=C1C(C=1C(=CC(=CC=1)S([O-])(=O)=O)S(O)(=O)=O)=C1C=CC(=[N+](CC)CC)C=C1 IKHKJYWPWWBSFZ-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000222178 Candida tropicalis Species 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
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- 108010006785 Taq Polymerase Proteins 0.000 description 1
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- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/72—Candida
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Abstract
The invention relates to candida nucleic acid extraction lysate, a kit and an extraction method. The cracking liquid comprises cracking liquid A, cracking liquid B and cracking liquid C; the pyrolysis liquid A is 0.20-0.30 mol/L of methylparaben solution; the lysate B is: 0.15 to 0.25mol/L lithium sulfate; the lysate C comprises: 40-60 mmol/L CTAB, 0.8-0.9 mol/L NaCl, 9-11 mmol/L EDTA, 90-110 mmol/L Tris-HCl. The pyrolysis liquid provided by the invention takes the methylparaben and the lithium sulfate as main active ingredients for the cell wall of the candida pyrolysis, lithium ions can change the charged property of the cell wall and the cell membrane surface, and the methylparaben and the lithium sulfate are combined to have the effect of efficiently pyrolyzing the cell wall and the cell membrane of the candida pyrolysis. In addition, CTAB in the lysate has the functions of denaturing protein and precipitating polysaccharide, and realizes one-step wall breaking, membrane breaking, protein and polysaccharide precipitation, so that the effects of releasing, dissociating and purifying candida genome DNA are achieved.
Description
Technical Field
The invention relates to candida nucleic acid extraction lysate, a kit and an extraction method, belonging to the technical fields of biomedical detection and bioengineering.
Background
Candida is a common pathogenic fungus, and comprises candida glabrata, candida tropicalis, candida krusei, candida albicans and the like, and can cause skin and mucous membrane infection of a human body, even cause invasive candidiasis such as pneumonia, eubacteremia and the like; the incidence rate of the mucous membrane infection of the skin is high, the mortality rate of severe patients is high, and the life and health of human beings are seriously threatened. Therefore, early diagnosis, rapid diagnosis, and accurate treatment have a great influence on improving the therapeutic effect and the quality of life of the patient. The traditional morphological identification method has the defects of long culture time and uncertain strain identification result, and is clinically used for diagnosis at present based on PCR amplification and genome sequencing analysis technologies. Efficient and safe extraction of pathogenic candida genomic DNA is the primary and key step in performing PCR amplification, genomic sequencing, and the like.
Candida has a cell wall which is solid, tough and complex in composition, so that effective disruption of the cell wall is a key to successful extraction of candida genomic DNA. In order to facilitate the cell wall cleavage and disruption, a repeated freeze thawing method, an ultrasonic disruption method, an alkaline disruption method, an enzymatic digestion method, and the like are currently used.
However, the above methods all have significant drawbacks: the repeated freezing and thawing method can be completed within 9-10 hours through refrigerator freezing, and is long in time consumption, short in time consumption but extremely poor in safety through liquid nitrogen freezing, and the wall breaking degree in the repeated freezing and thawing process is difficult to control, so that the cracking effect is poor, time and labor are wasted, and clinical requirements are difficult to meet. Although the ultrasonic disruption method is short in time consumption and only needs about 0.5h, the disruption is difficult to thoroughly, the extraction efficiency is extremely low, the DNA yield is low, and the clinical requirement is difficult to meet. The alkaline lysis method is quick in lysis, only about 0.5 hour is needed, but the extracted DNA is extremely easy to be seriously denatured, and downstream experiments cannot be carried out after the denaturation. Although the enzyme digestion method is not long in time consumption, generally about 1.5 hours, and not low in purity and yield, the method is very complicated in steps, high in requirements on instruments and extraction environment, and very expensive in price, and cannot be used in a large-scale and industrialized manner. Therefore, the existing candida genome DNA extraction method can not meet the requirements of rapid, batch, low cost and high-quality extraction of clinical samples, so that the method for extracting the candida nucleic acid rapidly and economically is provided, and the establishment of an efficient and simple candida nucleic acid extraction method still belongs to the technical problem.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides candida nucleic acid extraction lysate, a kit and an extraction method.
The technical scheme of the invention is as follows:
a candida nucleic acid extraction lysate comprises lysate A, lysate B and lysate C;
the pyrolysis liquid A is 0.20-0.30 mol/L of methylparaben solution;
the lysate B is: 0.15 to 0.25mol/L lithium sulfate;
the lysate C comprises: 40-60 mmol/L CTAB, 0.8-0.9 mol/L NaCl, 9-11 mmol/L EDTA, 90-110 mmol/L Tris-HCl.
According to the invention, preferably, the lysate A is 0.25mol/L of methylparaben solution; the pyrolysis liquid B is 0.2mol/L lithium sulfate; the lysate C comprises: 50mmol/L CTAB, 0.85mol/L NaCl, 10mmol/L EDTA, 100mmol/L Tris-HCl.
According to the invention, the lysate A is prepared according to the following method:
adding methylparaben into absolute ethyl alcohol according to the proportion, stirring until the methylparaben is fully dissolved, adding distilled water, stirring and uniformly mixing to obtain a pyrolysis liquid A, sealing, and preserving at room temperature in a dark place.
According to the invention, the lysate B is prepared according to the following method:
adding lithium sulfate into distilled water according to a proportion, stirring until the lithium sulfate is dissolved, adding distilled water, stirring and uniformly mixing to obtain a pyrolysis liquid B, sealing and preserving at room temperature.
According to the invention, the lysate C is preferably prepared as follows:
adding EDTA into distilled water according to the proportion, fully stirring at 55-65 ℃ until the EDTA is dissolved, then adding CTAB, fully stirring until the CTAB is dissolved, and cooling to room temperature to obtain component A; adding Tris into distilled water, adding NaCl, stirring until the Tris is fully dissolved, and adding concentrated hydrochloric acid to adjust the pH value to 8.0 to obtain a component B; adding the component B into the component A, adding distilled water, stirring and mixing uniformly to obtain a pyrolysis liquid C, sealing and preserving at room temperature.
A method for extracting candida nucleic acid by using the lysate comprises the following steps:
(1) Uniformly mixing the pyrolysis liquid A, the pyrolysis liquid B and the pyrolysis liquid C according to the volume ratio of 1:1 (4-16) to obtain pyrolysis liquid;
(2) Adding a lysis solution into a candida sample to be extracted, uniformly mixing, and incubating for 12-18 minutes at 50-60 ℃ to obtain a lysis mixture;
(3) Centrifuging the cleavage mixture at 12000 Xg for 5min, taking the supernatant, adding a nucleic acid sedimentation aid, uniformly mixing, centrifuging at 12000 Xg for 2min, discarding the supernatant, adding TE buffer solution into the precipitate to dissolve the precipitate, and obtaining the candida genome DNA.
According to the invention, in the step (1), the volume ratio of the lysate A, the lysate B and the lysate C is 1:1:10.
According to the invention, in the step (1), the volume-mass ratio of the lysate to the candida samples to be extracted is (0.6-0.8): 10, units: mL/mg.
According to a preferred embodiment of the present invention, in step (3), the nucleic acid precipitation aid is isopropanol precooled at-20 ℃.
According to a preferred embodiment of the invention, in step (3), the TE buffer composition comprises 1mmol/L EDTA and 10mmol/L Tris-HCl (pH 8.0).
The invention also provides a candida nucleic acid extraction kit which comprises the candida nucleic acid extraction lysate.
The beneficial effects of the invention are as follows:
1. the pyrolysis liquid provided by the invention takes the methylparaben and the lithium sulfate as main active ingredients for the cell wall of the candida pyrolysis, lithium ions can change the charged property of the cell wall and the cell membrane surface, and the methylparaben and the lithium sulfate are combined to have the effect of efficiently pyrolyzing the cell wall and the cell membrane of the candida pyrolysis. In addition, cetyl Trimethyl Ammonium Bromide (CTAB) in the lysate has the functions of denaturing protein and precipitating polysaccharide, and realizes one-step wall breaking, membrane breaking, protein and polysaccharide precipitation, so that the effects of releasing, dissociating and purifying candida genome DNA are achieved. And the components of the lysate are simple, the extraction cost is greatly reduced, and the lysate has wide application prospects in the field of microorganism detection.
2. The extraction method provided by the invention has the advantages of simple operation steps, strong repeatability and high extraction speed, can complete the whole extraction process only by 20min, avoids the pollution of human genes and environmental strain genes caused by complicated steps, and is suitable for batch and rapid operation of clinical samples.
Description of the drawings:
FIG. 1 is a graph showing agarose gel electrophoresis results of Candida genomic DNA extracted by the methods described in examples 1 to 3 and comparative examples 1 to 8 of the present invention: in the figure, M is Marker (200-1500 bp), A1-A3 and A4-A11 are Candida glabrata genome DNA extracted by the methods of examples 1-3 and comparative examples 1-8 in sequence.
FIG. 2 is a graph showing agarose gel electrophoresis results of Candida genomic DNA extracted according to the methods of examples 1 to 3 and comparative examples 1 to 8 of the present invention: in the figure, M is Marker (200-1500 bp), and B1-B3 and B4-B11 are Candida krusei genomic DNA extracted by the methods of examples 1-3 and comparative examples 1-8 in sequence.
FIG. 3 is a graph showing agarose gel electrophoresis results of Candida genomic DNA extracted according to the methods of examples 1 to 3 and comparative examples 1 to 8 of the present invention: in the figure, M is Marker (200-1500 bp), C1-C3 and C4-C11 are Candida albicans genome DNA extracted by the methods of examples 1-3 and comparative examples 1-8 in sequence.
FIG. 4 is a graph showing the result of agarose gel electrophoresis of PCR amplification using the Candida genomic DNA extracted by the methods described in examples 1 to 3 and comparative examples 1 to 8 of the present invention as a template: in the figure, M is Marker (200-1500 bp), C is blank control, A1-A3 and A4-A11 are candida glabrata genome DNA templates extracted by the methods of examples 1-3 and comparative examples 1-8 respectively.
FIG. 5 is a graph showing the result of agarose gel electrophoresis of PCR amplification using the Candida genomic DNA extracted by the methods of examples 1 to 3 and comparative examples 1 to 8 of the present invention as a template: in the figure, M is Marker (200-1500 bp), C is blank, and B1-B3 and B4-B11 are Candida krusei genome DNA templates extracted by the methods of examples 1-3 and comparative examples 1-8, respectively.
FIG. 6 is a graph showing the result of agarose gel electrophoresis of PCR amplification using the Candida genomic DNA extracted by the methods of examples 1 to 3 and comparative examples 1 to 8 of the present invention as a template: in the figure, M is Marker (200-1500 bp), C is blank control, and C1-C3 and C4-C11 respectively use Candida albicans genome DNA extracted by the methods of examples 1-3 and comparative examples 1-8 as templates.
Detailed Description
The invention will be further described with reference to specific examples for a better understanding of the invention, but the scope of the invention is not limited thereto. Meanwhile, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials, unless otherwise specified, are commercially available.
The test samples of Candida albicans, candida glabrata and Candida krusei in the following examples are all existing strains, and the concentration of each test sample is adjusted to 5×10 after the test sample is cultured in a Saccharopolyster agar medium and an enrichment medium 8 And taking 1mL of bacterial liquid per mL, and centrifugally collecting bacterial precipitate to be used as candida samples to be tested, wherein the bacterial content of each sample to be tested is consistent.
Example 1
A candida nucleic acid extraction lysate comprises lysate A, lysate B and lysate C;
the pyrolysis liquid A is 0.25mol/L of methylparaben solution;
the pyrolysis liquid B is 0.2mol/L lithium sulfate;
the lysate C comprises: 50mmol/L CTAB, 0.85mol/L NaCl, 10mmol/L EDTA, 100mmol/L Tris-HCl.
The lysate A is prepared according to the following method:
3.8038g (0.025 mol) of methylparaben is added into 75mL of absolute ethyl alcohol, stirred until the methylparaben is fully dissolved, 25mL of distilled water is added, stirred and mixed uniformly to obtain a lysate A, and the lysate A is stored in a dark place at room temperature after being sealed.
The lysate B is prepared according to the following method:
21.9880g (0.02 mol) of lithium sulfate was added to 75mL of distilled water, and the mixture was dissolved by stirring, and distilled water was added to a volume of 100mL.
The lysate C is prepared according to the following method:
3.3621g (10 mmol) of disodium ethylenediamine tetraacetate (EDTA) was added to 600mL of distilled water, stirred at 60℃until it was sufficiently dissolved, then 18.223g (50 mmol /) of CTAB was added, stirred sufficiently until it was dissolved and cooled to room temperature to obtain component A; 12.1130g (100 mmol) of Tris (hydroxymethyl) aminomethane (Tris) and 49.6740g (0.85 mol) of NaCl are added into 200mL of distilled water, stirred until the NaCl is fully dissolved, and concentrated hydrochloric acid is added to adjust the pH to 8.0, so that a component B is obtained; slowly adding the component B into the component A, continuously stirring during the period, requiring no obvious precipitation, adding distilled water to a volume of 1000mL to obtain a lysate C, sealing, and preserving at room temperature.
Candida albicans, candida glabrata and candida krusei are taken as candida samples to be extracted, and nucleic acid is respectively extracted by using the candida nucleic acid extraction lysate of the embodiment, and the specific steps are as follows:
(1) Before use, uniformly mixing the cracking liquid A, the cracking liquid B and the cracking liquid C according to the volume ratio of 1:1:10 to obtain a cracking working solution;
(2) Placing 10mg of candida samples to be extracted into an EP tube, adding 700 mu L of lysate, uniformly mixing, and incubating at 55 ℃ for 15 minutes to obtain a lysis mixture;
(3) The lysis mixture was centrifuged at 12000 Xg for 5 minutes, the supernatant was transferred to a clean EP tube, 1 volume of pre-chilled isopropanol at-20℃was added, gently and thoroughly mixed, the nucleic acid was precipitated, centrifuged at 12000 Xg for 2 minutes, the supernatant was discarded, and 50. Mu.L of TE buffer was added to the precipitate to dissolve the precipitate, thereby obtaining Candida genomic DNA.
Example 2
A candida nucleic acid extraction lysate, the raw material composition of which is described in example 1. Candida albicans, candida glabrata and candida krusei are taken as samples to be extracted, and nucleic acid is extracted by using the candida nucleic acid extraction lysate of the embodiment, wherein the specific steps are the same as those of the embodiment 1, and the specific steps are as follows: the volume mixing ratio of the lysate A, the lysate B and the lysate C is 1:1:4.
Example 3
A candida nucleic acid extraction lysate, the raw material composition of which is described in example 1. Candida albicans, candida glabrata and candida krusei are taken as samples to be extracted, and nucleic acid is extracted by using the candida nucleic acid extraction lysate of the embodiment, wherein the specific steps are the same as those of the embodiment 1, and the specific steps are as follows: the volume mixing ratio of the lysate A, the lysate B and the lysate C is 1:1:16.
Comparative example 1
A method for extracting candida nucleic acid, the extracted sample, the lysate used and the extraction steps are the same as in example 1, except that the mixing volume ratio of the lysate A, the lysate B and the lysate C is 1:1:1.
Comparative example 2
A method for extracting candida nucleic acid, the extracted sample, the lysate used and the extraction steps are the same as in example 1, except that the mixing volume ratio of the lysate A, the lysate B and the lysate C is 1:1:20.
Comparative example 3
A candida nucleic acid extraction lysate, the raw material composition of which is as described in example 1, except that lysate A does not contain methylparaben.
A method for extracting nucleic acid of Candida, nucleic acid is extracted by using the nucleic acid extracting lysate of Candida of this example, and the extracted sample and the specific procedure are the same as in example 1.
Comparative example 4
A candida nucleic acid extraction lysate, the raw material composition of which is as described in example 1, except that lysate B does not contain lithium sulfate.
A method for extracting nucleic acid of Candida, nucleic acid is extracted by using the nucleic acid extracting lysate of Candida of this example, and the extracted sample and the specific procedure are the same as in example 1.
Comparative example 5
A candida nucleic acid extraction lysate, the raw material composition of which is as described in example 1, except that lysate A does not contain methylparaben and lysate B does not contain lithium sulfate.
A method for extracting nucleic acid of Candida, nucleic acid is extracted by using the nucleic acid extracting lysate of Candida of this example, and the extracted sample and the specific procedure are the same as in example 1.
Comparative example 6
A method for extracting fungal genome DNA: adding 1000 mu L of sterile isotonic sodium chloride solution into a candida sample to be tested, freezing for 120min at the temperature of minus 20 ℃, thawing at room temperature, and repeating the freezing and thawing for 3 times; centrifuging at 12000 Xg for 10min, and discarding supernatant; adding 700 mu L of extracting solution, mixing by vortex, and incubating for 15min at 55 ℃; centrifuging 12000 Xg of the cracking mixture for 3min, transferring the supernatant to a clean EP tube, adding 1 time of pre-cooled isopropanol at the temperature of minus 20 ℃ and fully and uniformly mixing; 12000 Xg was centrifuged for 2min, the supernatant was discarded, and 50. Mu.L of TE buffer was added to dissolve the nucleic acid pellet.
The extracting solution comprises the following components: 2mol/L NaOH,0.5mol/L EDTA,2% SDS,100mmol/L Tris-HCl (pH 8.0).
Comparative example 7
A method for extracting fungal genome DNA: adding 1000 mu L of sterile isotonic sodium chloride solution into a candida sample to be tested, mixing uniformly by vortex, carrying out microwave high-temperature heating for 30s, cooling for 30s, and repeating for 4 times; centrifuging at 12000 Xg for 10min, and discarding supernatant; adding 700 mu L of extracting solution, mixing by vortex, and incubating for 15min at 55 ℃; centrifuging 12000 Xg of the cracking mixture for 5min, transferring the supernatant to a clean EP tube, adding 1 time of pre-cooled isopropanol at the temperature of minus 20 ℃ and fully and uniformly mixing; 12000 Xg was centrifuged for 5min, the supernatant was discarded, and 50. Mu.L of TE buffer was added to dissolve the nucleic acid pellet.
The extracting solution comprises the following components: 2mol/L NaOH,0.5mol/L EDTA,2% SDS,100mmol/L Tris-HCl (pH 8.0).
Comparative example 8
A method for extracting fungal genome DNA: the method uses an Shanghai engineering Ezup column type yeast genome DNA extraction kit (B518257), the kit adopts a snail enzyme method to crack fungus cell walls, and the operation method refers to a product instruction book and comprises the following specific steps: 600 mu LSnailase Reaction Buffer is added into a candida sample to be tested, 2.4 mu L of beta-mercaptoethanol and 50 mu LsNAILase are added, and water bath is carried out for 3 hours at 37 ℃; centrifuging at 12000 Xg for 2min at room temperature, and discarding supernatant; 200 mu L Buffer Digestion is added, 20 mu L of protease K solution is added, and the mixture is vibrated and mixed uniformly; water bath at 56 ℃ for 1h until the cells are completely lysed; adding 100 μl Buffer PY, mixing, standing at-20deg.C for 5min; centrifuge at 12000 Xg for 5min at room temperature, transfer supernatant into new EP tube; 200 mu L Buffer BD is added, and the mixture is fully inverted and uniformly mixed; adding 200 mu L of absolute ethyl alcohol, fully reversing and uniformly mixing; placing the adsorption column into a collecting pipe, adding the solution and semitransparent fibrous suspension into the adsorption column, standing for 2min, centrifuging at 12000 Xg at room temperature for 1min, and discarding the waste liquid; placing the adsorption column into a collecting tube, adding 500 μl of PW Solution, centrifuging for 30s at 12000×g, and discarding the waste liquid; placing the adsorption column into a collecting pipe, adding 500 μl of Wash Solution, centrifuging for 30s at 12000×g, and discarding the waste liquid; putting the adsorption column back into the collection tube again, centrifuging at 12000 Xg room temperature for 2min, and leaving residual Wash Solution; the column was taken out and put into a new EP tube, 50. Mu.L of TE Buffer was added thereto and allowed to stand still for 3min,12000 Xg was centrifuged at room temperature for 2min, and the DNA solution was collected.
Experimental example 1
The genomic DNA concentrations and purities of examples 1 to 3 and comparative examples 1 to 6 were measured using a Nanodrop micro nucleic acid protein concentration meter, and the results are shown in Table 1 below.
As is clear from Table 1, the use of the lysate of example 1 can efficiently lyse common candida such as Candida glabrata, candida krusei, candida albicans, etc., and fully release nucleic acids, and the genomic DNA concentration obtained by the extraction method of example 1 reaches 322.4-450.2 ng/. Mu.L, and the purity is high, A 260nm /A 280nm 1.9. As is clear from the results of comparative examples 1 to 2 and comparative example 1, when the mixing volume ratio of lysate C in the lysate is too low, the extracted genome concentration approaches that of example 1, but the purity is low, A 260nm /A 280nm About 1.7, indicating protein residues; as is clear from the results of comparative examples 1, 3 and comparative example 2, when the mixing volume ratio of the lysate C in the lysate is too high, the efficiency of lysing the cells is significantly lowered,the yield of extracted genomic DNA was too low.
The results of comparative examples 3-5 show that the lysate does not contain methylparaben and lithium sulfate (comparative example 5), the concentration of the extracted genomic DNA is only about 20 ng/. Mu.L, and no obvious lysis effect is achieved; when the lysate contains lithium sulfate alone (comparative example 3) or methylparaben (comparative example 4), the concentration of the extracted genomic DNA does not exceed 100 ng/. Mu.L; when the methylparaben and the lithium sulfate are combined, the concentration of the extracted nucleic acid is obviously higher than that of the single overlapped nucleic acid, and the methylparaben and the lithium sulfate have obvious synergistic effect.
The genomic DNA concentrations of comparative example 6 and comparative example 7 were significantly lower than those of the methods of the present invention, indicating that the conventional repeated freeze thawing and sonication methods have low fungus cell lysis efficiency. The genome DNA concentration of the test fungi provided by the Shanghai snail enzyme method kit (comparative example 8) is 133.6-168.3 ng/. Mu.L, which is lower than that of example 1, A 260nm /A 280nm The purity is about 1.9, but the extraction time is longer than 90min, and the extraction operation steps are up to 12 steps. The extraction time of the embodiment 1 only needs 20min, the extraction of the genomic DNA of the sample can be completed, the time is far lower than that of a snail enzyme method kit, the concentration is about 1 time higher, the cost is far lower than that of a comparative example 8 kit, and the technical advantage is obvious.
Experimental example 2
Agarose gel electrophoresis detection is carried out on candida genomic DNA extracted in examples 1 to 3 and comparative examples 1 to 8, and the specific method is as follows: mu.L of genomic DNA was mixed with 1. Mu.L of 6×loading buffer, and 1% agarose gel electrophoresis was performed with 5. Mu.L of each sample Loading, and the results are shown in FIGS. 1 to 3.
1-3, the Candida glabrata, candida krusei and Candida albicans genomic DNA electrophoresis strips extracted in example 1 are clear and bright, and have no obvious dispersion and tailing; the brightness of the strips of the mixture ratio of the lysate A, the lysate B and the lysate C is similar to that of the embodiment 1, which shows that the better cracking effect can be achieved by increasing the concentration of the methylbenzoate and the lithium sulfate in the lysate, which is consistent with the ultraviolet detection result, but the purity of the extracted nucleic acid is reduced and the cost of the reagent is increased; when the mixing proportion is too high, the concentration of the methylparaben and the lithium sulfate in the lysate is low, and the obtained band is fuzzy, which indicates that the DNA content of the sample is low; the brightness of the strips of the comparative examples 3-4 is obviously lower than that of the example 1, and 5 strips of the comparative example are not obvious, which shows that the combination of the methylparaben and the lithium sulfate in the lysate can obviously improve the cell wall lysis effect. The brightness of the strips of comparative example 6 and comparative example 7 was much lower than in example 1 of the method of the invention, and the snail enzyme kit of comparative example 8 was similar to that of example 1, but with slightly lower brightness.
Experimental example 3
The fungus 18S rDNA sequences were amplified using the Candida genomic DNA extracted in examples 1 to 3 and comparative examples 1 to 8 as templates, and the primers were synthesized by Shanghai, and the sequences were as follows:
an upstream primer: 5'-GCATCGATGAAGAACGCAGC-3';
a downstream primer: 5'-TCCTCCGCTTATTGATATGC-3'.
The PCR reaction system is as follows:
the 2 XPCR mix is a common commercial product, and comprises the main components: 100mM KCl, 3mM MgCl 2 0.4mM dNTP mix, 0.1U/. Mu.L Taq DNA polymerase, 20mM Tris-HCl (pH 8.3), xylene blue and orange dye.
PCR reaction procedure: pre-denaturation at 94℃for 3min; denaturation at 94℃for 30s, annealing at 55℃for 45s, elongation at 72℃for 1min,30 cycles; extending at 72℃for 3min. The detection results of the PCR amplification products by agarose gel electrophoresis are shown in FIGS. 4-6.
As can be seen from FIGS. 4 to 6, the amplified bands of Candida krusei, candida glabrata and Candida albicans extracted in example 1, example 2 and comparative example 1 were bright and clear, and the bands obtained in comparative example 5 were slightly lower in brightness without nonspecific amplified bands, and the three Candida nucleic acid templates of other groups did not amplify the obvious target fragments. The candida genome DNA extracted by the lysate and the extraction method provided by the invention can meet the downstream PCR amplification requirement.
In conclusion, the pyrolysis liquid provided by the invention creatively uses the methylparaben and the lithium sulfate as main active ingredients of the candida pyrolysis liquid, and the effects of efficiently cracking candida cell walls and cell membranes are found after the methylparaben and the lithium sulfate are combined, and then the effects of breaking walls, breaking membranes, precipitating proteins and polysaccharides in one step are achieved by combining components such as CTAB (CTAB), so that nucleic acid is fully dissociated, the efficient extraction of candida genome DNA is realized, and the whole extraction process can be completed only by 20 min. The extraction method provided by the invention solves the problem of low efficiency of cracking candida cell walls in the traditional methods such as repeated freeze thawing method, ultrasonic crushing method, alkaline cracking method and the like. Compared with the enzyme method kit, the method can obtain the genome DNA with the concentration 1 time higher than that of the enzyme method kit by only about 20 minutes in the extraction process, has obviously higher extraction efficiency and has far lower cost than that of the enzyme method; and the complicated operation steps of the extraction process are greatly simplified, the batch, rapid and safe operation of samples can be realized, the method is suitable for clinic, the extracted samples can stably amplify candida 18S rDNA fragments, and powerful support can be provided for the subsequent identification of strains by combining with a sequencing technology.
Claims (10)
1. The candida nucleic acid extraction lysate is characterized by comprising a lysate A, a lysate B and a lysate C;
the pyrolysis liquid A is 0.20-0.30 mol/L of methylparaben solution;
the lysate B is: 0.15 to 0.25mol/L lithium sulfate;
the lysate C comprises: 40-60 mmol/L CTAB, 0.8-0.9 mol/L NaCl, 9-11 mmol/L EDTA, 90-110 mmol/L Tris-HCl.
2. The candida nucleic acid extraction lysate of claim 1 in which lysate a is a 0.25mol/L solution of methylparaben; the pyrolysis liquid B is 0.2mol/L lithium sulfate; the lysate C comprises: 50mmol/L CTAB, 0.85mol/L NaCl, 10mmol/L EDTA, 100mmol/L Tris-HCl.
3. The candida nucleic acid extraction lysate of claim 1, wherein lysate a is formulated as follows:
adding methylparaben into absolute ethyl alcohol according to the proportion, stirring until the methylparaben is fully dissolved, adding distilled water, stirring and uniformly mixing to obtain a pyrolysis liquid A, sealing, and preserving at room temperature in a dark place.
4. The candida nucleic acid extraction lysate of claim 1, wherein lysate B is formulated as follows:
adding lithium sulfate into distilled water according to a proportion, stirring until the lithium sulfate is dissolved, adding distilled water, stirring and uniformly mixing to obtain a pyrolysis liquid B, sealing and preserving at room temperature.
5. The candida nucleic acid extraction lysate of claim 1, wherein lysate C is formulated as follows:
adding EDTA into distilled water according to the proportion, fully stirring at 55-65 ℃ until the EDTA is dissolved, then adding CTAB, fully stirring until the CTAB is dissolved, and cooling to room temperature to obtain component A; adding Tris into distilled water, adding NaCl, stirring until the Tris is fully dissolved, and adding concentrated hydrochloric acid to adjust the pH value to 8.0 to obtain a component B; adding the component B into the component A, adding distilled water, stirring and mixing uniformly to obtain a pyrolysis liquid C, sealing and preserving at room temperature.
6. A method for extracting candida nucleic acid using the lysate of claim 1, comprising the steps of:
(1) Uniformly mixing the pyrolysis liquid A, the pyrolysis liquid B and the pyrolysis liquid C according to the volume ratio of 1:1 (4-16) to obtain pyrolysis liquid;
(2) Adding a lysis solution into a candida sample to be extracted, uniformly mixing, and incubating for 12-18 minutes at 50-60 ℃ to obtain a lysis mixture;
(3) Centrifuging the cleavage mixture at 12000 Xg for 5min, taking the supernatant, adding a nucleic acid sedimentation aid, uniformly mixing, centrifuging at 12000 Xg for 2min, discarding the supernatant, adding TE buffer solution into the precipitate to dissolve the precipitate, and obtaining the candida genome DNA.
7. The method of claim 6, wherein in step (1), the volume ratio of lysate A, lysate B, and lysate C is 1:1:10.
8. The method of claim 6, wherein in step (1), the ratio of the volume to mass of the lysate to the candida sample to be extracted is (0.6-0.8): 10, units: mL/mg.
9. The method of claim 6, wherein in step (3), the nucleic acid precipitation aid is pre-chilled isopropyl alcohol at-20 ℃; the TE buffer component contained 1mmol/L EDTA and 10mmol/L Tris-HCl (pH 8.0).
10. A candida nucleic acid extraction kit, characterized in that the kit comprises the candida nucleic acid extraction lysate of claim 1.
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