CN114480371A - Method for extracting microbial DNA from sample - Google Patents
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- CN114480371A CN114480371A CN202210083547.7A CN202210083547A CN114480371A CN 114480371 A CN114480371 A CN 114480371A CN 202210083547 A CN202210083547 A CN 202210083547A CN 114480371 A CN114480371 A CN 114480371A
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Abstract
A method for extracting microbial DNA from a sample comprises the main processes of sample liquefaction and pathogenic microbial nucleic acid extraction, wherein the liquefaction comprises the addition of a liquefaction reagent and Benzonase nuclease, the Benzonase nuclease is added at the same time, so that the sample can be doubly liquefied, part of host nucleic acid can be removed, the extraction time of a metagenome is reduced, and the subsequent separation of the pathogenic microbial DNA from a clinical sample is facilitated.
Description
Technical Field
The invention relates to the field of microbial molecular detection, in particular to a method for extracting microbial DNA from a sample, wherein the main sample comprises sputum and abscess fluid.
Background
The infection caused by respiratory diseases mainly relates to sputum samples, common infectious pathogenic microorganisms of the sputum samples comprise acinetobacter baumannii, streptococcus pneumoniae, mycobacterium tuberculosis, moraxella catarrhalis, pseudomonas aeruginosa, aspergillus fumigatus, aspergillus niger herpes simplex virus and the like, the pathogenic microorganisms are infectious and need to be strictly operated in a biosafety cabinet, and a common laboratory does not have operation equipment. The conventional sputum sample is viscous, the mucin in the sputum sample can be destroyed only by liquefaction treatment, the currently most applied liquefaction reagents are sodium hydroxide, dithiothreitol and N-acetic acid-L cysteine, and in 2009 military force and other people, four sputum treatment methods are compared to find that the saccomanno method and the DTT method are good in sputum fixation and liquefaction, but the treatment methods are long in time, host nucleic acid cannot be removed, pathogenic microorganism treatment time is prolonged, and experimental data analysis difficulty is increased. In the aspect of respiratory tract microorganisms, the progress is slow because the respiratory tract samples have high viscosity, more colonized bacteria and high content of human-derived nucleic acid and pathogenic microorganisms are difficult to judge.
The abscess is generated in a plurality of parts and can be positioned on the body surface or in the abdominal cavity, staphylococcus aureus is a common pathogenic bacterium causing the abscess, but the pathogenic bacteria causing the abscess at different parts are quite different, and sometimes the pathogenic bacterium causing infection needs to be found out by other means after the medicine is ineffective. The traditional pathogen detection methods include smear microscopy, immunological detection, microbial culture, and molecular detection. However, these methods can only detect one or a limited number of pathogens at a time, and in addition, the culture has the disadvantage of low positive rate, and most of abscess solution collected in clinical samples is relatively viscous, the whole samples are cloudy, precipitation and separation after high-speed centrifugation are not obvious, the supernatant is not easy to be removed completely, and the obtained nucleic acid product extracted from the precipitate has low purity and is mostly host nucleic acid. The emerging metagenome detection technology (mNGS) can sequence the microbial population genome in a specific environment, avoids the defect that most of microbes cannot be cultured in the traditional method, and brings difficulty for detecting pathogenic microbes with high human DNA ratio.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for efficiently extracting pathogenic microorganism DNA from a sample, which not only fully liquefies the sample, but also adds a human source nucleic acid removing reagent, thereby reducing the time cost, increasing the extraction space of pathogenic microorganisms and providing favorable conditions for the extraction of the metagenome of the pathogenic microorganisms.
In order to achieve the purpose, the invention is realized by the following scheme: a method of extracting microbial DNA from a sample, comprising the steps of:
(1) adding a liquefaction reagent and a reagent for removing a host into the sample box, and performing double liquefaction, superposition and host removal process on the sample;
the liquefaction reagent comprises a methanol solution and a dithiothreitol solution, and the volume ratio of the methanol solution to the dithiothreitol solution is 8-12: 1; the dithiothreitol solution is prepared by dissolving dithiothreitol in a methanol solution, wherein the concentration of the dithiothreitol is 0.08-0.12g/mL, and the mass fraction of the methanol solution is 10% -30%;
the nuclease in the agent for removing the host is
A nuclease;
the components of the nuclease lysate are Tris-Hcl and Mgcl;
(2) after incubation for half an hour at 37 ℃, adding a reaction stopping reagent to quench the reaction, wherein the reaction stopping reagent comprises EDTA and NaCl; EDTA final concentration of 3-5mM, pH 7.0-8.0; the final concentration of NaCl is 140-150 mM;
(3) centrifuging after full oscillation, discarding the supernatant, adding PBS for washing, centrifuging again, discarding the supernatant, and adding PBS to obtain a treated sample;
(4) breaking microbial cells, and extracting microbial nucleic acid; microbial cells are crushed by a boiling method, a grinding method or a chemical cracking method, and DNA is extracted by a column passing method after the crushing.
The boiling method adopts a physical heating method to break microbial cells; the grinding method belongs to a mechanical method for breaking pathogenic microorganism cells, and adopts zirconium oxide beads as grinding beads; the chemical cracking method belongs to a chemical method for breaking pathogenic microorganism cells, and adopts chemical reagents of proteinase K and lysate. The sample is a sputum sample or an abscess fluid sample. Firstly, a double liquefaction and superposition host removing process of a sample; ② extracting nucleic acid of pathogenic microorganism; the efficient liquefaction for the sample also has the effect of removing part of host nucleic acid, and is used for extracting pathogenic microorganism nucleic acid after removing part of the host nucleic acid. The sample liquefaction process comprises liquefaction reagents and nucleases: the liquefaction reagent comprises: methanol, dithiothreitol; the concentration of the liquefied reagent methanol is 10% -30%; the preparation of the liquefaction reagent dithiothreitol is that a certain amount of dithiothreitol is weighed, 10 to 30 percent of methanol reagent is added, and the mixture is put into a refrigerator for standby after being shaken up;
the reagent used in the host removal process is nuclease; the nuclease is
Nuclease (5 KU); off-host process in order to
The nuclease needs to be prepared and used by itself
A nuclease lysate; the main components of the zymolysis solution are Tris-Hcl and Mgcl;
the reaction terminating reagent used in the host removing process is EDTA and NaCl; the final concentration of the termination reagent EDTA is 3-5mM, and the pH value is 7.0-8.0; the final concentration of NaCl is 140-150 mM;
the extraction process of pathogenic microorganism nucleic acid has boiling process, and belongs to physical method for breaking pathogenic microorganism cells;
boiling process in the extraction process of pathogenic microorganism nucleic acid, wherein the boiling temperature is 90-95 ℃; the pathogenic microorganism nucleic acid extraction process has a grinding process and belongs to the technical field of breaking pathogenic microorganism cells by a physical heating method;
grinding in the pathogenic microorganism nucleic acid extraction process, wherein the grinding beads are zirconia beads; the extraction process of pathogenic microorganism nucleic acid has a chemical cracking process, and belongs to the technical field of breaking pathogenic microorganism cells by a chemical method;
the pathogenic microorganism nucleic acid extraction process is to extract DNA by a column-crossing method.
The invention has the beneficial effects that: adding a liquefaction reagent (comprising methanol DTT,
Nuclease) the sputum became significantly homogeneous at this point
The nuclease plays double roles, on one hand, the nuclease promotes the homogenization of sputum, on the other hand, the nuclease digests nucleic acid exposed by incomplete cells (host cells have no cell wall structure and are easier to break), and the measurement of the concentration of a sample is reflected,
the addition of nuclease can reduce the extraction concentration of the sample to a certain extent, on the other hand, the proportion of the original DNA of the sample can be reduced, and the proportion of pathogenic microorganisms is improved to a certain extent.
The method can more efficiently and more sufficiently reduce the viscosity of the sample, more sufficiently homogenize the sputum sample, more obviously separate the supernatant of the abscess fluid sample, remove the nucleic acid pollution of damaged host cells in the sample, greatly save the time cost, and is favorable for the rapid detection of the clinical pathogenic microorganism metagenome of the sample.
Detailed Description
Example 1 sputum sample
To better illustrate that the simultaneous addition of the liquefaction reagent and the Benzonase nuclease can reduce the viscosity of the sample to make the sample more homogeneous and can also reduce the proportion of the sample to the original DNA, comparative example 1 is provided, namely, no Benzonase nuclease is added in comparative example 1.
In example 1: directly adding 5 times volume of 30% methanol solution and prepared DTT solution into the sputum sample box at a ratio of 10:1 (such as 1000ul methanol +100ul DTT) in a clean bench; after the membrane is sealed, the sample is shaken until no viscous substance is visible to naked eyes, and the sample is subpackaged into a centrifugal tube of 1.5 ml; 1ul of Benzonase enzyme and 10 XBenzonase buffer was added to 1.5ml of sample to a final concentration of 1X. Benzonase buffer includes Tris-HCl and Mgcl.
Incubation was carried out at 37 ℃ for half an hour, and the reaction was quenched by adding EDTA (final concentration 5mM, pH 8.0) and NaCl (final concentration 150mM) to the tube;
centrifuging 8000g for 10min after sufficient shaking, discarding the supernatant, adding PBS for washing, centrifuging 8000g for 10min, discarding the supernatant, adding 500ul PBS;
winding sealing film, and clamping with explosion-proof clamp, and incubating at 95 deg.C for 10 min. Transferring into a grinding tube for grinding; the nucleic acid is extracted by a column chromatography method by using the kit.
In comparative example 1: directly adding 5 times volume of 30% methanol solution and prepared DTT solution into the sputum sample box at a ratio of 10:1 (such as 1000ul methanol +100ul DTT) in a clean bench; after the membrane is sealed, the sample is shaken until no viscous substance is visible to naked eyes, and the sample is subpackaged into a centrifugal tube of 1.5 ml;
centrifuging 1500g for 10min, removing supernatant, washing with 1ml PBS, centrifuging 1500g for 10min, removing supernatant, and adding 500ul PBS;
winding sealing film, and clamping with explosion-proof clamp, and incubating at 95 deg.C for 10 min. Transferring into a grinding tube for grinding; the nucleic acid is extracted by a column chromatography method by using the kit.
The experimental results are as follows:
observing the experimental results, it can be seen that the extracted concentration of the sample in comparative example 1 (i.e. without adding Benzonase nuclease) is generally higher than that in example 1, because the DNA in comparative example 1 contains most of the host DNA, while the addition of the liquefaction reagent and Benzonase nuclease in example 1 can make the sample homogenized more sufficiently, remove most of the host nucleic acid, have obviously reduced variation in concentration, and obviously reduce the human source proportion% of the sample, and the reduced human source difference range is 2.81% -11.73%, which is more beneficial to the subsequent microbial data analysis. This example illustrates the simultaneous addition of a liquefaction reagent (including methanol DTT) and a destroing reagent to a sputum sampleReagent-
The nuclease can obviously homogenize the sputum
The nuclease also plays another role in digesting the exposed nucleic acid of incomplete cells (host cells have no cell wall structure and are easy to break), reduces the extraction concentration of a sample and the proportion of the original DNA of the sample, improves the proportion of pathogenic microorganisms to a certain extent, can extract the nucleic acid of the microorganisms more efficiently compared with the traditional treatment method, and is beneficial to the rapid detection of clinical sample pathogenic microorganism metagenome.
Example 2 abscess solution
To better illustrate that the simultaneous addition of the liquefaction reagent and the Benzonase nuclease can not only make the separation of the abscess liquid supernatant from the sediment more obvious, but also reduce the proportion of the original DNA of the sample, a comparative example 2 is specially arranged, namely no Benzonase nuclease is added in the comparative example 2.
In example 2: adding 4 times volume of 30% methanol solution and prepared DTT solution into the tube for collecting abscess solution at a ratio of 12:1 (such as 1200ul methanol +100ul DTT) in a clean bench; after the membrane is sealed, the sample is shaken until no viscous substance is visible to naked eyes, and the sample is subpackaged into 2.0ml centrifugal tubes; to 1.5ml of sample, add 1ul of Benzonase enzyme (250U) and 10x Benzonase buffer to a final concentration of 1 x; benzonase buffer includes Tris-HCl and Mgcl.
Incubation was carried out at 37 ℃ for half an hour, and the reaction was quenched by adding EDTA (final concentration 5mM, pH 8.0) and NaCl (final concentration 150mM) to the tube; centrifuging 8000g for 10min after sufficient oscillation, discarding the supernatant, adding 1ml PBS for washing, centrifuging 8000g for 10min, discarding the supernatant, adding 500ul PBS;
winding sealing film, clamping with explosion-proof clamp, and incubating at 95 deg.C for 10 min; transferring the liquid in the pipe into a grinding pipe for grinding; and (4) extracting nucleic acid from the sampled and ground sample by a column chromatography method by using the kit.
In comparative example 2: adding 4 times volume of 30% methanol solution and prepared DTT solution into the tube for collecting abscess solution at a ratio of 12:1 (such as 1200ul methanol +100ul DTT) in a clean bench; after the membrane is sealed, the sample is shaken until no viscous substance is visible to naked eyes, and the sample is subpackaged into 2.0ml centrifugal tubes;
centrifuging at 16000g for 10min, removing supernatant, adding 500ul PBS into the precipitate, and resuspending the sample; winding sealing film, and clamping with explosion-proof clamp, and incubating at 95 deg.C for 10 min. Transferring into a grinding tube for grinding; the nucleic acid is extracted by a column chromatography method by using the kit.
The experimental results are as follows:
observing the experimental results of the abscess solution, it can be seen that the sample extraction concentration in comparative example 2 (i.e. without adding Benzonase nuclease) is generally higher than that in example 2, because the DNA in comparative example 2 contains most of host DNA, while the addition of the liquefaction reagent and Benzonase nuclease in example 2 can make the sample homogenate more sufficient, the separation of supernatant and precipitate is obvious, the purity of the nucleic acid extracted from the sample meets the standard, most importantly, most of the host nucleic acid is removed, the human source ratio% of the sample is reduced, the human source difference range is reduced to 2.73% -7.05%, and the subsequent analysis of microorganism data is facilitated. This example demonstrates that the use of a liquefaction reagent and Benzonase nuclease in an abscess fluid sample can effectively reduce the viscosity of the abscess fluid sample, and that the reduction of the host nucleic acid ratio in the sample is more beneficial to the extraction of microbial DNA.
Claims (3)
1. A method for extracting microbial DNA from a sample, comprising the steps of:
(1) adding a liquefaction reagent and a reagent for removing a host into the sample box, and performing double liquefaction, superposition and host removal process on the sample;
the liquefaction reagent comprises a methanol solution and a dithiothreitol solution, and the volume ratio of the methanol solution to the dithiothreitol solution is 8-12: 1; the dithiothreitol solution is prepared by dissolving dithiothreitol in a methanol solution, wherein the concentration of the dithiothreitol is 0.08-0.12g/mL, and the mass fraction of the methanol solution is 10% -30%;
the nuclease in the agent for removing the host is
A nuclease;
the components of the nuclease lysate are Tris-Hcl and Mgcl;
(2) after incubation for half an hour at 37 ℃, adding a reaction stopping reagent to quench the reaction, wherein the reaction stopping reagent comprises EDTA and NaCl; EDTA final concentration of 3-5mM, pH 7.0-8.0; the final concentration of NaCl is 140-150 mM;
(3) centrifuging after full oscillation, discarding the supernatant, adding PBS for washing, centrifuging again, discarding the supernatant, and adding PBS to obtain a treated sample;
(4) breaking microbial cells, and extracting microbial nucleic acid; microbial cells are crushed by a boiling method, a grinding method or a chemical cracking method, and DNA is extracted by a column passing method after the crushing.
2. The method of claim 1, wherein the step of extracting the DNA of the microorganism comprises: the boiling method adopts a physical heating method to break microbial cells; the grinding method belongs to a mechanical method for breaking pathogenic microorganism cells, and adopts zirconium oxide beads as grinding beads; the chemical cracking method belongs to a chemical method for breaking pathogenic microorganism cells, and adopts chemical reagents of proteinase K and lysate.
3. The method of claim 1, wherein the step of extracting the DNA of the microorganism from the sample comprises: the sample is a sputum sample or an abscess fluid sample.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115678966A (en) * | 2022-11-14 | 2023-02-03 | 北京金匙医学检验实验室有限公司 | Multi-sample type universal metagenome rapid detection method |
| CN115873670A (en) * | 2022-11-29 | 2023-03-31 | 苏州近岸蛋白质科技股份有限公司 | Biological enzyme scavenger for eliminating nucleic acid pollution and preparation method thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949748A (en) * | 2018-07-30 | 2018-12-07 | 浙江今复康生物科技有限公司 | A kind of sputum liquefaction and nuclease protection reagent |
| CN111088249A (en) * | 2020-01-19 | 2020-05-01 | 泰州健为医学检验实验有限公司 | Use method of metagenome sample de-hosting extraction kit |
| CN111621496A (en) * | 2020-05-06 | 2020-09-04 | 南京世和基因生物技术股份有限公司 | Sputum metagenome de-hosting extraction kit |
| CN112063617A (en) * | 2020-10-09 | 2020-12-11 | 亚能生物技术（深圳）有限公司 | Method and kit for rapidly extracting tubercle bacillus nucleic acid from sputum |
| CN113575573A (en) * | 2021-09-07 | 2021-11-02 | 易普森生物科技(深圳)有限公司 | Preservation solution for sputum sample and application thereof |
-
2022
- 2022-01-25 CN CN202210083547.7A patent/CN114480371A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949748A (en) * | 2018-07-30 | 2018-12-07 | 浙江今复康生物科技有限公司 | A kind of sputum liquefaction and nuclease protection reagent |
| CN111088249A (en) * | 2020-01-19 | 2020-05-01 | 泰州健为医学检验实验有限公司 | Use method of metagenome sample de-hosting extraction kit |
| CN111621496A (en) * | 2020-05-06 | 2020-09-04 | 南京世和基因生物技术股份有限公司 | Sputum metagenome de-hosting extraction kit |
| CN112063617A (en) * | 2020-10-09 | 2020-12-11 | 亚能生物技术（深圳）有限公司 | Method and kit for rapidly extracting tubercle bacillus nucleic acid from sputum |
| CN113575573A (en) * | 2021-09-07 | 2021-11-02 | 易普森生物科技(深圳)有限公司 | Preservation solution for sputum sample and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 张军力;托娅;王育民;: "不同的液化方法对痰液DNA提取效果比较", 检验医学, no. 06, pages 456 - 458 * |
| 文岚: "5种前处理方法对痰中结核分枝杆菌DNA提取的影响", 实用预防医学, vol. 20, no. 09, pages 307 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115678966A (en) * | 2022-11-14 | 2023-02-03 | 北京金匙医学检验实验室有限公司 | Multi-sample type universal metagenome rapid detection method |
| CN115873670A (en) * | 2022-11-29 | 2023-03-31 | 苏州近岸蛋白质科技股份有限公司 | Biological enzyme scavenger for eliminating nucleic acid pollution and preparation method thereof |
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