CN114561380A - Bacterial nucleic acid extraction detection reagent, kit, method and application thereof - Google Patents
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Abstract
The invention provides a detection reagent for extracting bacterial nucleic acid, a kit, a method and an application thereof, wherein the detection reagent for extracting bacterial nucleic acid contains InstaGeneMatrix, SDS, KCl, EDTA and Sarkosyl; coli nucleic acid can be rapidly and efficiently extracted from a complex sample, and the potential clinical applicability of the extraction method is verified.
Description
Technical Field
The invention relates to the technical field of biology, in particular to the technical field of extraction and detection of bacterial nucleic acid.
Background
Nucleic acid is a biological macromolecular compound formed by polymerizing a plurality of nucleotides, and is one of the most basic substances of life. Nucleic acid extraction refers to a process of separating nucleic acid from a sample by physical and chemical methods, and the separation of high-quality nucleic acid is a key step in molecular biology research. The conventional methods for extracting nucleic acid include conventional phenol-chloroform method, salting-out method, membrane centrifugation column method, etc. The nucleic acid extraction methods widely applied by a silica gel membrane adsorption column method, an immune affinity method of an anti-DNA monoclonal antibody and an automatic platform are provided by commercialized nucleic acid extraction kits as the current mainstream nucleic acid extraction methods. However, the conventional nucleic acid extraction kit has poor quality of extracted nucleic acid and cannot meet the actual demand.
In recent years, with the development of molecular biology and biotechnology, detection technologies based on nucleic acid amplification are emerging, such as Real-time polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP) and metagenomic sequencing (mNGS), which have the characteristics of rapidness and sensitivity, and have been widely used to determine the presence of escherichia coli in clinical samples. However, the detection process still has the defects of complex operation, more introduced reagents and less relative content of bacteria, so that the content of effective substances in the DNA lysate is low. For example, a special silicon substrate material is adopted to efficiently and specifically adsorb nucleic acid under the conditions of certain high salt and low pH value and release nucleic acid under the conditions of low salt and high pH value; or a magnetic bead purification method based on separation and purification of reversible immobilization of a solid phase carrier is widely applied to the fields of nucleic acid extraction, purification and the like. However, the two nucleic acid extraction techniques are complicated in steps, and especially, the pretreatment step involves multiple liquid phase transfer and solution adding processes, so that the operation time of the whole process is long. Not only is cross contamination easy to cause, but also the cost is higher, so that the cost of large-scale wide application is high.
Disclosure of Invention
Aiming at one of the technical problems recorded in the content, the invention provides a bacterial nucleic acid extraction and detection reagent, a kit, a method and application thereof, which improve the current embarrassment of bacterial nucleic acid extraction and detection, reduce the operation steps and greatly reduce the possibility of cross contamination. Compared with two common methods mentioned in the background art, the method also reduces the use cost, reduces the use cost to one two hundredth and one fiftieth of the original use cost respectively compared with a column method and a magnetic bead method, has the detection limit lower than 100 copies/ml, and is superior to most detection platforms in the market at present.
In a first aspect, the invention provides a kit for extracting and detecting bacterial nucleic acid, which comprises a primer sequence and an extraction detection reagent, wherein the extraction detection reagent consists of InstaGene Matrix with the mass concentration of 1% -5%, SDS with the mass concentration of 1% -20%, KCl with the mass concentration of 10mmol/L-100mmol/L, EDTA with the mass concentration of 1mmol/L-10mmol/L, and Sarkosyl with the mass concentration of 1% -5%.
In a second aspect, the invention provides an application of the bacterial nucleic acid extraction and detection kit in escherichia coli nucleic acid extraction and detection.
Specifically, the sequence information of the primers used for extraction and detection in the invention is as follows: SEQ-ID NO 1: TGCGGGCGGCTTT, respectively; SEQ-ID NO 2: GGCGGCGCAAAAA are provided.
In a third aspect, the present invention also provides a method for determining the extraction limit of bacterial nucleic acid, the method comprising the steps of:
(1) setting a combination of a plurality of different microorganisms and adding a human background, wherein the input parameter of bacteria is 8-15,000 copy numbers/ml;
(2) nucleic acid extraction: centrifuging and precipitating the prepared 1ml of mixed bacteria liquid with the parameter of 10000-; adding 200 mul of nucleic acid extracting solution to bacterial colony sediment, shaking and mixing evenly; then placing the mixture in a water bath at 100 ℃, standing for 5-10 minutes, and then cooling for 1-3 minutes at room temperature; centrifuging and precipitating the cracking product at a high speed with the parameter of 10000-; taking 50-100 mul of supernatant for subsequent quality control and target gene detection;
(3) and (3) concentration measurement and quality control: using NanoReady to carry out quality control on the extracted nucleic acid solution, wherein the parameter is A260/A280; meanwhile, the Agilent2100 is adopted to carry out quality control on the extracted nucleic acid;
(4) extraction of the control method group: extracting according to a column method;
(5) and (3) comparing extraction results: the comparative parameters include extraction time, extraction cost, quality control of extracted nucleic acid, total extraction amount and PCR amplification detection result;
(6) PCR detection and verification: the method comprises PCR amplification detection of simulated mixed sample nucleic acid and second-generation metagenome sequencing mNGS verification, wherein the second-generation metagenome sequencing mNGS verification comprises breaking and library building, on-machine sequencing and data analysis, and the extraction limit of extracting the escherichia coli nucleic acid is determined.
Specifically, the PCR amplification of the invention adopts a common PCR mix kit;
the PCR detection and verification of the invention is to carry out agarose gel electrophoresis analysis and subsequent second-generation sequencing analysis on the amplification product.
In a fourth aspect, the invention provides a bacterial nucleic acid extraction detection reagent, which consists of InstaGene Matrix, SDS, KCl, EDTA and Sarkosyl.
In a fifth aspect, the invention provides a bacterial nucleic acid extraction detection reagent, wherein the mass concentration of the InstaGene Matrix in the extraction detection reagent is 1% -5%; the mass concentration of SDS is 1% -20%; the mass concentration of KCl substance is 10mmol/L-100 mmol/L; the mass concentration of EDTA substance is 1mmol/L-10mmol/L, and the mass concentration of Sarkosyl is 1% -5%; the extraction detection reagent is a mixed solution which is prepared by mixing the InstaGene Matrix, SDS, KCl, EDTA and Sarkosyl to form corresponding concentration.
In a sixth aspect, the invention provides a bacterial nucleic acid extraction and detection method, which comprises the following steps:
(1) centrifuging and precipitating the sample containing the nucleic acid with the parameter of 10000-;
(2) adding a nucleic acid extraction detection reagent consisting of InstaGene Matrix, SDS, KCl, EDTA and Sarkosyl into the colony precipitate obtained in the step (1), and shaking and uniformly mixing; then placing the mixture in a boiling water bath, standing for 5-10 minutes, and then cooling for 1-3 minutes at room temperature;
(3) centrifuging and precipitating the cracking product at a high speed, wherein the parameter is 10000-;
(4) and taking the supernatant for subsequent quality control and target gene detection.
Specifically, the extraction detection method further includes: the primer design and synthesis, PCR amplification, PCR detection and result analysis.
Specifically, the sequence information of the designed and synthesized primer is as follows: SEQ-ID NO 1: TGCGGGCGGCTTT, respectively; SEQ-ID NO 2: GGCGGCGCAAAAA are provided.
By implementing the technical scheme of the invention, the following beneficial effects can be achieved:
the reagent, the kit and the method for extracting and detecting the bacterial nucleic acid can realize the rapid and efficient extraction of the bacterial nucleic acid, and the cost is reduced to one two hundredth of that of a column method and one fiftieth of that of a magnetic bead method through the embodiment verification.
Drawings
FIG. 1 is a flow chart of nucleic acid extraction in the present method;
FIG. 2 is a gel electrophoresis image of bacteria detection.
Legend: detecting the specific gene of the escherichia coli by gel electrophoresis; m and DNAmarker are 2000bp, 1500bp, 1000bp, 750bp, 500bp, 250bp and 100bp from top to bottom in sequence; CK is DNA extracted from a control group, and T1-3 is DNA extracted by the method; the results show that the amplification efficiency of the phoA gene in the nucleic acid extracted by the method is obviously better than that of the nucleic acid of the control group compared with the control group.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the drawings in the specification, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention will be further understood by reference to the following detailed description of preferred embodiments of the invention and the examples included therein.
Coli used in the present invention was purchased from Dowabo bioengineering, Inc. in mountain. 1ml of sterilized normal saline is used for preparing suspension bacteria liquid, Qiagen nucleic acid extraction kit (cargo number: 56204) is used for extracting the bacterial nucleic acid by a column method, then spectrophotometry is used for quantifying, the total mass of the nucleic acid which can be extracted by each tube is verified, genome copy number calculation is carried out by taking the total mass as a standard, and a reference line is established for the subsequent extraction detection limit.
The microorganism of the invention is combined into a commercial reagent ZymobiomicsTMThe Microbial Community Standard (product number: D6300, hereinafter referred to as "Zymo") includes ten kinds of common microorganismsBiological, using parameters of 1.4x106cell/ml; a human HeLa cell line (purchased from North Nay Bio) with the use parameter of 1.0x10 was also added5cell/ml; the input parameter of the escherichia coli is 8-16,000 copy number/ml; the interval is set for detecting the detection limit of extraction, the dilution multiple is 5, and the middle escherichia coli input parameters are 40, 200, 1000 and 5000 copy numbers/ml respectively.
The comparison group compared with the extraction method of the invention carries out nucleic acid detection according to column extraction, and the embodiment of the comparison group of the invention selects a Qiagen nucleic acid extraction kit to carry out column extraction of the bacterial nucleic acid.
The first embodiment is as follows: bacterium nucleic acid extraction detection reagent
The preparation method of the solution comprises the following steps: mixing InstaGene Matrix (1-5%, w/v, Bio-Rad), SDS (1-20%, w/v), KCl (10-100mM), EDTA (1-10mM) and Sarkosyl (1-5%, w/v); mixing; the buffer solution is prepared, autoclaved and subpackaged at 4 ℃ for long-term storage for later use.
Example two: reagent for extracting and detecting bacterial nucleic acid
The kit is used for extracting and detecting the bacterial nucleic acid, and comprises an extraction detection reagent, wherein the extraction detection reagent contains InstaGene Matrix with the mass concentration w/v of 1-5%; SDS with the mass concentration w/v of 1-20%; the mass concentration of the substance is 10mmol/L-100mmol/L KCl; substances are mixed with EDTA in a concentration of 1mmol/L to 10mmol/L and Sarkosyl in a concentration w/v of 1% to 5%.
Example three: method for extracting escherichia coli nucleic acid from nucleic acid extracting solution
The nucleic acid extracting solution is used for extracting the nucleic acid of escherichia coli, and comprises the following specific steps:
a: centrifuging and precipitating the prepared 1ml of mixed bacterium liquid at 12000rpm for 1 min, and removing the supernatant;
b: adding 200 mul of nucleic acid extracting solution to bacterial colony sediment, shaking and mixing evenly; then placing the mixture in a water bath at 100 ℃, standing for 8 minutes, and then cooling for 1-3 minutes, preferably 2 minutes at room temperature;
c: centrifuging the cracked product at high speed for precipitation, wherein the parameter is 12000rpm and 2 minutes;
d: taking 100 mu l of supernatant for subsequent quality control and target gene detection;
the quality control of nucleic acid, in this example, NanoReady (Hangzhou kansui) was used to control the quality of the extracted nucleic acid solution, with the parameters of A260/A280 and A260/A230 nucleic acid concentration; and simultaneously, the Agilent2100 is adopted to carry out quality control on the extracted nucleic acid.
Example four: method for extracting Escherichia coli nucleic acid from nucleic acid extracting solution
The nucleic acid extracting solution is used for extracting the nucleic acid of escherichia coli, and comprises the following specific steps:
a: centrifuging and precipitating the prepared 1ml of mixed bacterium liquid with the parameter of 10000rpm for 1 minute, and removing the supernatant;
b: adding 200 mul of nucleic acid extracting solution to the colony sediment, shaking and mixing uniformly; then the mixture was placed in a water bath at 100 ℃ for 5 minutes, and then cooled at room temperature for 1 minute;
c: centrifuging the cracked product at high speed for 2 minutes with the parameter of 10000 rpm;
d: taking 50 mu l of supernatant for subsequent quality control and target gene detection;
the quality control of nucleic acid, in this example, NanoReady (Hangzhou kansui) was used to control the quality of the extracted nucleic acid solution, with the parameters of A260/A280 and A260/A230 nucleic acid concentration; meanwhile, the Agilent2100 is adopted to carry out quality control on the extracted nucleic acid;
example five: method for extracting escherichia coli nucleic acid from nucleic acid extracting solution
The nucleic acid extracting solution is used for extracting the nucleic acid of escherichia coli, and comprises the following specific steps:
a: centrifuging and precipitating the prepared 1ml of mixed bacteria liquid with the parameter of 14000rpm for 1 minute, and removing the supernatant;
b: adding 200 mul of nucleic acid extracting solution to the colony sediment, shaking and mixing uniformly; then placing the mixture in a water bath at 100 ℃, standing for 10 minutes, and then cooling for 3 minutes at room temperature;
c: centrifuging and precipitating the lysate at high speed with the parameter of 14000rpm for 2 minutes;
d: taking 150 mu l of supernatant for subsequent quality control and target gene detection;
the quality control of nucleic acid, in this example, NanoReady (Hangzhou kansui) was used to control the quality of the extracted nucleic acid solution, with the parameters of A260/A280 and A260/A230 nucleic acid concentration; and simultaneously, the Agilent2100 is adopted to carry out quality control on the extracted nucleic acid.
The quality control information of the fourth to sixth examples is shown in the following table:
the treatment groups are T1-3 which respectively correspond to the nucleic acid quality control information extracted by the methods of the fourth to sixth embodiments, the comparison groups are CK 1-3 which are the same in composition of the treatment groups, and the nucleic acid quality control information is respectively extracted by adopting a standard column method.
Results and discussion: compared with the control group for extraction time, extraction cost, quality control of extracted nucleic acid, total extraction amount, PCR amplification detection results and the like, the cost of the method is reduced to one two hundredth of that of a column method and one fiftieth of that of a magnetic bead method, the quality control standard of the extracted nucleic acid meets the downstream detection requirements, and the method can be used for efficiently extracting escherichia coli in simulation and metagenome samples.
Example six: method for extracting nucleic acid and detecting escherichia coli by using nucleic acid extracting solution
In this example, a complex sample set was used to perform nucleic acid extraction, PCR amplification, PCR detection and result analysis of E.coli.
(1) Extracting nucleic acid of escherichia coli by using the nucleic acid extraction detection reagent of the embodiment 1 and the extraction method of the embodiment 3;
(2) designing and synthesizing primers: the sequence information of the primers designed and synthesized in this example is: SEQ-ID NO 1: TGCGGGCGGCTTT; SEQ-ID NO 2: GGCGGCGCAAAAA, respectively; finally, dissolving the synthesized primer by using enzyme-free water, wherein the final concentration is 10 mu M;
(3) and (3) PCR amplification: adopting a common PCR mix kit, preferably adopting domestic Novovovovozam Taq Mastermix (Dye Plus); the PCR amplification system is as follows:
| components | Volume (μ L) |
| Taq Master Mix(2X) | 10 |
| DNA template | 5 |
| Forward primer (10. mu.M) | 0.5 |
| Reverse primer (10. mu.M) | 0.5 |
| DNase/RNase-Free water | Upto25 |
| Total | 25 |
The PCR amplification procedure was as follows:
(4) PCR detection and verification: carrying out agarose gel electrophoresis analysis and subsequent second-generation sequencing analysis on the amplification product; the electrophoresis condition parameters are as follows: 120V, 30 minutes, preferably a domestic six-brand horizontal electrophoresis apparatus; the sample application amount is 5 mu L/lane;
(5) results and discussion: through PCR amplification and electrophoretic analysis, the extraction method provided by the invention is determined to be capable of effectively extracting Escherichia coli under simulated and complex backgrounds.
Example seven: detection of Escherichia coli in mixed microorganism strains by using next-generation metagenomic sequencing (mNGS)
The method comprises the following specific steps:
(1) breaking and building a library: the operation steps provided by the kit are adopted to interrupt and build the library by adopting a domestic Novovzan second-generation library building kit (the cargo number is ND 617);
(2) and (3) machine sequencing: adopting an Illumina computer-on sequencing kit (cargo number: 20024906), and utilizing a kit recommendation operation instruction to complete computer-on sequencing;
(3) and (3) data analysis: and (3) after quality control and human source removal are carried out on the off-line data, reads of TB specificity and other species are identified by using Blast, and finally identification information is output in batches.
Example eight: method for determining extraction limit of Escherichia coli nucleic acid
In this embodiment, the extraction limit of the escherichia coli nucleic acid is preliminarily explored by setting the escherichia coli input amount with different copy numbers, and the specific steps are as follows:
(1) setting a complex sample group: the main difference lies in the difference of the copy number of the added inactivated escherichia coli; the copy number of Escherichia coli put into the experiment of this example is 102，103And 104Copy number/ml; background organisms include ten common microorganisms contained in Zymo, using parameters of 1.4X106cell/ml; meanwhile, a humanized HeLa cell strain is added, and the use parameter is 1.0x105cell/ml；
(2) The method provided by the embodiment of the invention is used for extracting the nucleic acid of the sample, and performing concentration measurement and quality control;
(3) the control method group adopts column method extraction, preferably a Qiagen nucleic acid extraction kit for control test;
(4) carrying out mNGS sequencing verification on the detection of the nucleic acid of the simulated mixed sample according to the seventh step embodiment, and determining the extraction limit of the method for extracting the nucleic acid of the escherichia coli;
results and discussion: as can be determined from the data in the table below, three different copy numbers (10) can be extracted by the method of the invention2，103And 104Copy number/ml) of E.coli nucleic acid, and the number of reads after second-generation sequencing was kept at 102Above, it was demonstrated that the method can be used to extract E.coli nucleic acids in a complex sample background.
Example nine: performance comparison of nucleic acid extraction and detection reagents for different bacteria
In the embodiment of the present invention, different combinations of reagents for extracting and detecting bacterial nucleic acid were used to extract the complex sample set of the previous embodiment of the present invention by the method of embodiment 3, and the results of the complex sample set were compared with the data of the baseline 9 established by using the detection limit of escherichia coli, and the following results were obtained
Shown in Table 3:
| processing number | Name of process | Performance enhancement (fold; Total DNA yield/treatment 9) |
| 1 | Buffer1(InstaGene+SDS+KCl+EDTA+Sarkosyl) | 10±0.44 |
| 2 | Buffer2(InstaGene+SDS+EDTA+Sarkosyl) | 5.4±0.24 |
| 3 | Buffer3(InstaGene+SDS+Sarkosyl) | 5.7±0.15 |
| 4 | Buffer4(InstaGene+Sarkosyl) | 7.1±0.24 |
| 5 | Buffer5(InstaGene+KCl) | 5.8±0.24 |
| 6 | Buffer6(SDS+KCl+EDTA+Sarkosyl) | 6.1±0.39 |
| 7 | Buffer7(InstaGene+EDTA) | 4.5±0.24 |
| 8 | Buffer8(InstaGene+KCl+EDTA) | 5.6±0.35 |
| 9 | Qiagen kit | 1.0±0.17 |
In the test of taking the detection reagent, the mass concentration of the selected InstaGene Matrix is 2%, the mass concentration of SDS is 10%, the mass concentration of KCl substance is 50mmol/L, the mass concentration of EDTA substance is 5mmol/L, and the mass concentration of Sarkosyl is 2%. The action and the mutual relation of the substances are judged by continuously increasing and decreasing the substance detection and comparing with the data which are already published and reported by the substance.
As can be seen from the above data and the published data of the above substances, the reagent for extracting and detecting bacterial nucleic acid provided by the embodiment has the best effect, and the extraction performance is not influenced by the uniform degree of one or more components; meanwhile, the different components are in certain correlation, wherein the action proportion of the detergent and the chelating agent accounts for a higher level, repeated experiments in the embodiment show that the substances not only have the functions of breaking cells, chelating divalent ions of EDTA, buffering and the like, but also have influence on other reagents, and experiments in the invention show that the combination of InstaGene, SDS, KCl, EDTA and Sarkosyl has a good synergistic effect.
Example ten: performance comparison of nucleic acid extraction and detection reagents for different bacteria
In the embodiment of the present invention, the usage amounts of the substances in example 1 are adjusted, the method in example 3 is used to extract the complex sample set according to the embodiment of the present invention, and the results of the complex sample set are compared with the baseline established by using the escherichia coli extraction detection limit, and the obtained results are shown in table 4 below:
in the test of the detection reagent, different mass concentrations of InstaGene Matrix, KCl and EDTA and different mass concentrations of SDS and Sarkosyl are selected, wherein the numbers 1, 2, 3 and 4 are test groups, the numbers 5, 6, 7, 8, 9, 10, 11, 12, 13 and 14 are control groups with individual substance concentrations out of the range of the application, and 15 is a reference group. By comparing different amounts of substances, the invention finds that the combination of InstaGene, SDS, KCl, EDTA and Sarkosyl has a great influence on the result except for a very good synergistic effect, which is totally different from the expectation that the amount of each substance can be obtained by a single experiment, and the performance is obviously reduced after exceeding the limit of the concentration range of the invention, which should be that the equivalent is increased or reduced to a certain amount to influence other reagents.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Claims (10)
1. The kit for extracting and detecting the bacterial nucleic acid is characterized by comprising a primer sequence and an extraction detection reagent, wherein the extraction detection reagent consists of five reagents, namely InstaGene Matrix with the mass concentration of 1% -5%, SDS with the mass concentration of 1% -20%, KCl with the mass concentration of 10mmol/L-100mmol/L, EDTA with the mass concentration of 1mmol/L-10mmol/L and Sarkosyl with the mass concentration of 1% -5%.
2. The use of the kit for extracting and detecting bacterial nucleic acid according to claim 1 in extraction and detection of Escherichia coli nucleic acid.
3. The application of the kit for extracting and detecting bacterial nucleic acid in the extraction and detection of Escherichia coli nucleic acid as claimed in claim 1, wherein the primer sequence information adopted by the extraction and detection is as follows: SEQ-ID NO 1: TGCGGGCGGCTTT, respectively; SEQ-ID NO 2: GGCGGCGCAAAAA are provided.
4. A method for determining the limit of extraction of bacterial nucleic acid, said method comprising the steps of:
(1) setting a combination of various different microorganisms and adding a human-derived background, wherein the input parameter of escherichia coli is 8-15,000 copy number/ml;
(2) nucleic acid extraction: centrifuging and precipitating the prepared 1ml of mixed bacteria liquid with the parameter of 10000-; adding 200 mul of nucleic acid extracting solution to the colony sediment, shaking and mixing uniformly; then placing the mixture in a water bath at 100 ℃, standing for 5-10 minutes, and then cooling for 1-3 minutes at room temperature; centrifuging and precipitating the cracking product at a high speed with the parameter of 10000-; taking 50-100 mul of supernatant for subsequent quality control and target gene detection;
(3) and (3) concentration measurement and quality control: performing quality control on the extracted nucleic acid solution by using NanoReady, wherein the parameter is A260/A280; meanwhile, the Agilent2100 is adopted to carry out quality control on the extracted nucleic acid;
(4) extraction of the control method group: extracting according to a column method;
(5) and (3) comparing extraction results: the comparative parameters include extraction time, extraction cost, quality control of extracted nucleic acid, total extraction amount and PCR amplification detection result;
(6) PCR detection and verification: comprises PCR amplification detection and second-generation sequencing analysis of the nucleic acid of a simulated mixed sample, and determines the extraction limit of the extracted escherichia coli nucleic acid.
5. The method for determining the extraction limit of bacterial nucleic acid according to claim 4, wherein the PCR detection and verification comprises agarose gel electrophoresis analysis of the amplified product and second-generation metagenomic sequencing mNGS verification, and the second-generation metagenomic sequencing mNGS verification comprises breaking and banking, on-machine sequencing and data analysis.
6. A bacterial nucleic acid extraction detection reagent is characterized by consisting of InstaGene Matrix, SDS, KCl, EDTA and Sarkosyl.
7. The reagent of claim 6, wherein the InstaGene Matrix is present in an amount of 1% to 5% by weight of the reagent; the mass concentration of SDS is 1% -20%; the mass concentration of KCl substance is 10mmol/L-100 mmol/L; the mass concentration of EDTA substance is 1mmol/L-10mmol/L, and the mass concentration of Sarkosyl is 1% -5%; the extraction detection reagent is a mixed solution which is prepared by mixing the InstaGene Matrix, SDS, KCl, EDTA and Sarkosyl to form corresponding concentration.
8. A bacterial nucleic acid extraction and detection method is characterized by comprising the following steps:
(1) centrifuging and precipitating the sample containing the nucleic acid with the parameter of 10000-;
(2) adding a nucleic acid extraction detection reagent consisting of InstaGene Matrix, SDS, KCl, EDTA and Sarkosyl into the colony precipitate obtained in the step (1), and shaking and uniformly mixing; then placing the mixture in a boiling water bath, standing for 5-10 minutes, and then cooling for 1-3 minutes at room temperature;
(3) centrifuging and precipitating the cracking product at a high speed with the parameter of 10000-;
(4) and taking the supernatant for subsequent quality control and target gene detection.
9. The method for extracting and detecting bacterial nucleic acid according to claim 8, further comprising: the primer design and synthesis, PCR amplification, PCR detection and result analysis.
10. The method for extracting and detecting bacterial nucleic acid according to claim 9, wherein the sequence information of the designed and synthesized primer is: SEQ-ID NO 1: TGCGGGCGGCTTT, respectively; SEQ-ID NO 2: GGCGGCGCAAAAA are provided.
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