CN111041118A - Method for rapidly detecting flora structure in drainage and production water of coal-bed gas well by using PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) - Google Patents
Method for rapidly detecting flora structure in drainage and production water of coal-bed gas well by using PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular biology, and provides a method for rapidly detecting a flora structure in drainage and production water of a coal-bed gas well by using PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis) in order to solve the problem of how to rapidly detect and understand the dynamic change of the flora structure on each node in an experimental period. Anaerobic collection of drainage and sampling water samples is carried out on a drainage and sampling site of the coal bed methane well, the drainage and sampling water samples are used as a bacteria source, then a culture medium is added, anthracite retrieved from a drainage and sampling well is used as a substrate, and the anthracite is cultured in an anaerobic bottle; collecting culture solution at several nodes of a culture period of an anaerobic gas production experiment, extracting DNA, designing a pair of universal primers for PCR amplification by using a variable region of 16S rDNA V1-V3 bacteria, and separating and recovering fragments by DGGE electrophoresis; and (5) sequencing after the second amplification, and detecting the change of the flora structure in the drainage and production water of the coal-bed gas well. The method has low cost and easy operation, and can realize rapid detection of the variety of the gas-producing flora and the change of the flora structure in the anaerobic fermentation methane-producing experiment.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a method for rapidly detecting a flora structure in drainage and production water of a coal-bed gas well by using PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis).
Background
As a renewable clean energy source, the production of coal bed gas is expected to reach the target of 100 billionths of cubic meters. Biogenic gas is an important component of coal bed gas, and students have made more researches on the formation mechanism of biogenic gas, and prove that coal can be degraded by microorganisms and converted into biomethane under appropriate laboratory conditions. The coal microbial gasification technology mainly utilizes the action of microbes such as fungi, bacteria and actinomycetes to destroy the structure of coal molecules, so that the coal is liquefied to generate substances capable of being further metabolized, and finally biogas is generated. Therefore, the research on the microbial diversity of the coal bed is significant to the research and development of the biogenesis coal bed gas.
The development of modern molecular biology technology enables the technology to be widely applied to the research of the microbial structure of coal beds or coal bed water. Therefore, the method for rapidly detecting the change of the flora structure in the anaerobic fermentation gas production experiment process by using the coal bed water as the bacteria source by using the molecular biology method has important significance for the basic research of the whole coal microorganism gas production technology.
Disclosure of Invention
In an anaerobic fermentation methane production experiment taking drainage and production water of a coal bed gas well as a bacterial source, the flora structure in the coal bed water in an anaerobic reactor in an experiment period changes, and how to quickly detect and know the dynamic change of the flora structure on each node in the experiment period becomes an urgent problem to be solved; in order to solve the problems, the invention provides a method for rapidly detecting the flora structure in drainage and production water of a coal-bed gas well by using PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis), and the flora structure in a coal biological gas formation experiment can be rapidly detected by using a molecular biology technology so as to realize the understanding and regulation of the flora structure.
The invention is realized by the following technical scheme: a method for rapidly detecting the flora structure in drainage and production water of a coal bed gas well by utilizing PCR-DGGE (polymerase chain reaction-denaturing gradient gel electrophoresis), wherein a drainage and production water sample is anaerobically collected at a drainage and production site of the coal bed gas well, stored at 4 ℃ in a laboratory by using site liquid nitrogen, taken as a bacterial source, added with a culture medium, and cultured in an anaerobic bottle by using anthracite retrieved from the drainage and production well as a substrate; collecting culture solution at several nodes of a culture period of an anaerobic gas production experiment, extracting DNA, designing a universal primer by using a variable region of bacteria 16S rDNA V1-V3 for PCR amplification, and separating and recovering fragments by DGGE electrophoresis; and (5) sequencing after the second amplification, and detecting the change of the flora structure in the drainage and production water of the coal-bed gas well.
The method comprises the following specific steps:
(1) culturing strains: taking a water sample collected from a coal bed well which is preserved in an anaerobic way as a bacteria source, adding a culture medium and a coal sample, and culturing in an anaerobic bottle; taking a 500ml anaerobic bottle, adding 25g of coal powder as a substrate in an anaerobic box, adding 250ml of culture medium and 50ml of water sample, sealing, and culturing in a constant-temperature incubator at 35 ℃;
(2) and (3) extracting DNA: collecting bacterial liquid at a plurality of time nodes in a culture period, filtering and collecting thalli, and extracting DNA and storing at-20 ℃;
(3) sequence amplification and purification: performing PCR sequence amplification twice and purifying:
for the first amplification, a pair of universal primers is designed according to a variable region of 16S rDNA V1-V3 of bacteria for PCR amplification, wherein the sequence of an upstream primer is 27F: 5-AGAGT TTGAT CCTGG CTCAG-3;
the downstream primer is 518R: 5-ATTAC CGCGG CTGCT GG-3;
the PCR amplification condition is pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 65 ℃ for 1min, extension at 72 ℃ for 1min, and 25 cycles; extending for 10min at 72 ℃;
for the second amplification, a pair of universal primers is designed according to the variable region of the bacteria 16S rDNA V3 for PCR, wherein a hairpin framework of 30-40bp is inserted into the 5' end of the upstream primer; the PCR upstream primer sequence is 338F-GC: 5-CGCCC GCCGC GCGCGGCGGG CGGGG CGGGG GCACG GGGGG ACTCC TACGG GAGGC AGCAG-3;
the sequence of the downstream primer is 518R: 5-ATTAC CGCGG CTGCT GG-3;
wherein the sequence of the GC hairpin structure is 5-CGCCC GCCGC GCGCG GCGGG CGGGG CGGGG-3;
the PCR amplification conditions comprise pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 1min, extension at 72 ℃ for 2min and 25 cycles; extending for 10min at 72 ℃;
detecting PCR amplification products by 2% gel electrophoresis;
(4) DGGE electrophoresis and imaging observations: the concentration of polyacrylamide gel is 10 percent, the gel denaturation gradient is 40 to 60 percent, and the Buffer solution is 1 multiplied by TAE Buffer; polymerizing the denatured gel for 90 minutes, then inserting a 16-comb into the space at the top of the denatured gel, adding 25 mu L of PCR product into each hole by using a sample injection needle, carrying out electrophoresis at the temperature of 60 ℃ and the voltage of 200V for 5min, and then carrying out electrophoresis at the temperature of 60 ℃ and the voltage of 85V for 12 h; then dyeing in 3 XGelRed dye for 25-35min, and observing gel imaging under an ultraviolet lamp of a gel irradiation instrument;
(5) cutting gel, recovering a target band, and sequencing: marking the clear common and specific bands in the DGGE map with cut gel, putting the cut gel into a 1.5mL centrifuge tube, adding 20 mu L deionized water, mashing the gel, dissolving the gel, standing overnight at 4 ℃, and sequencing a dissolving solution;
(6) analyzing a sequencing result: and performing comparison analysis, coumarone-Vera index analysis and sample uniformity analysis by using an NCBI Blast program to determine main variable strains of the bacterial liquid samples taken from a plurality of nodes.
The culture medium in the step (1) is an inorganic culture medium, and the formula of the culture medium is 2g/L K2HPO4、1.5g/L KH2PO4、0.4g/L MgCl2、1g/L NH4Cl, 2g/L YE; the coal sample is anthracite in a drainage and mining underground coal bed.
One culture period in the step (2) is 25-35 days; the plurality of time nodes are a gas production early stage, a gas production rising stage, a gas production stable stage and a gas production descending stage.
Comparing and analyzing the sequencing result by using an NCBI Blast program in the step (6) to determine common and specific strains in the flora structures of different samples; and obtaining the number, brightness and position specific data of the strips by contrasting the DGGE map, and obtaining the change condition of the flora structure in different samples.
The bands at different positions of each DGGE lane in the DGGE electrophoresis in the step (4) represent different bacteria, and the brightness of the bands reflects the relative amount of the bacteria.
Compared with the prior art, the flora structure in the coal bed water in the anaerobic reactor in an experimental period is changed continuously, the method can quickly and efficiently detect and know the dynamic change of the flora structure on each node in the experimental period, and can analyze the target sequence in a targeted manner. The existing high-throughput sequencing technology has the advantages that the cycle for testing a sample is about 30 days, the whole sequence is sequenced, and the cost is higher. The detection period of the method is 3 days, preliminary judgment can be made by observing lane strip changes, if large changes are found, gel cutting and sequencing analysis are performed in a targeted manner, multiple times of high-throughput sequencing can be avoided, and the method can be fast, efficient and cost-saving.
The invention can quickly and visually observe and detect the change condition of the flora structure in the samples at different periods. The cost is low, the operation is easy, and the rapid detection of the flora structure change among samples can be realized.
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FIG. 1 is a 1% agarose gel electrophoresis of extracted DNA, M is DL2000 DNA Marker; 1-4 is extracted DNA product;
FIG. 2 is a DGGE diagram of drainage and production water of a coal-bed gas well at different culture times;
FIG. 3 is a diagram showing the classification of bacteria in drainage and production water of a coal-bed gas well at a gate level by analyzing the sequencing result;
FIG. 4 is a rarity curve of bacteria in drainage and production water of a coal-bed gas well obtained by analyzing a sequencing result.
Detailed Description
In the embodiment, drainage and production water of the coal-bed gas well is used as a bacteria source, a proper amount of culture medium is added under the laboratory condition, anthracite retrieved from a drainage and production well is used as a substrate, and the anthracite is cultured in an anaerobic bottle. Collecting culture solution on different nodes of a culture period, and rapidly detecting the change of the flora structure in drainage and production water of the coal-bed gas well by a PCR-DGGE method. The flora structure in the coal bed water in the anaerobic reactor in an experimental period is changed continuously, the method can quickly and efficiently detect and know the dynamic change of the flora structure on each node in the experimental period, and the target sequence is analyzed in a targeted manner. The existing high-throughput sequencing technology has the advantages that the cycle for testing a sample is about 30 days, the whole sequence is sequenced, and the cost is higher. The detection period of the method is 3 days, preliminary judgment can be made by observing lane strip changes, if large changes are found, gel cutting and sequencing analysis are performed in a targeted manner, multiple times of high-throughput sequencing can be avoided, and the method can be fast, efficient and cost-saving. The method comprises the following specific steps:
(1) collecting and storing strains: and (3) anaerobically collecting drainage and sampling water samples by using an anaerobic bottle at a drainage and sampling site of the coal bed gas well, and storing the collected drainage and sampling water samples by using site liquid nitrogen and returning the collected drainage and sampling water samples to a laboratory for storage at 4 ℃ for later use.
(2) Samples were collected at different incubation time nodes and DNA was extracted: taking the retrieved and stored water sample as a bacteria source, adding a proper amount of culture medium, taking anthracite retrieved from a drainage well as a substrate, and culturing in an anaerobic bottle. Collecting bacterial liquid at different time nodes in a culture period, filtering and collecting thalli, extracting DNA by using a Water DNA kit (OMEGA) according to the standard operation of a specification, and detecting the purity and the content of the extracted DNA by using a nucleic acid protein instrument (Eppendorf); the value of A260/A280 is 1.8-2.0, which shows that the DNA extracted by the kit has high purity and is stored at the temperature of-20 ℃. The 1% agarose gel electrophoresis of the extracted DNA is shown in FIG. 1.
(3) Two PCR sequences were amplified and purified: the first amplification is carried out by designing a pair of universal primers for PCR amplification according to the variable region of the 16S rDNA (V1-V3) of bacteria, wherein the sequence of an upstream primer is 27F: 5-AGAGTTTGATCCTGGCTCAG-3;
the downstream primer is 518R: 5-ATTACCGCGGCTGCTGG-3. The PCR amplification condition is pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 65 ℃ for 1min, extension at 72 ℃ for 1min, and 25 cycles; extension at 72 ℃ for 10 min.
The second amplification is to design a pair of universal primers for PCR according to the variable region of the bacterial 16S rDNA V3, wherein the 5' end of the upstream primer is inserted with a hairpin structure of 30-40 bp. The PCR primers were 338F-GC: 5-CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGGGACTCCTACGGGAGGCAGCAG-3;
the downstream primer is 518R: 5-ATTACCGCGGCTGCTGG-3. Wherein the sequence of the GC hairpin structure is 5-CGCCC GCCGCGCGCG GCGGG CGGGG CGGGG-3. The PCR amplification conditions comprise pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 1min, extension at 72 ℃ for 2min and 25 cycles; extension at 72 ℃ for 10 min. Detecting PCR amplification products by 2% gel electrophoresis.
(4) DGGE electrophoresis and imaging observations: the concentration of polyacrylamide gel is 10%, the gel denaturation gradient is 40% -60%, and the Buffer solution is 1 × TAE Buffer. The denatured gel was polymerized for 90 minutes, then a 16-comb was inserted from the top gap of the denatured gel, 25. mu.L of PCR product was added to each well with a needle, and pre-electrophoresis was performed at 60 ℃ and 200V for 5min, and then electrophoresis was performed at 60 ℃ and 85V for 12 hours. Then dyeing in 3 XGelRed dye for about 30min, and observing gel imaging under an ultraviolet lamp of a gel irradiation instrument.
FIG. 2 shows DGGE graphs of drainage water of coal-bed gas wells at different culture times, wherein different position bands in each DGGE lane represent different bacteria, the more bands show that the flora composition is more complex, the brightness of the bands reflects the relative amount of the bacteria, and the higher the brightness shows that the proportion of the bacteria in the flora is larger.
(5) Cutting the gel and recovering the target band, and then sequencing: the clear common and specific bands in the DGGE spectrum are marked with tapping, 20 mu L of deionized water is added into a 1.5mL centrifuge tube, the gel is mashed by a gun head and stays overnight at 4 ℃, and the gel is dissolved as much as possible.
The lysates were then submitted for sequencing.
(6) Analyzing a sequencing result: determining the main variable strains of the bacterial liquid samples taken from different nodes. The analysis method comprises the following steps: the NCBI Blast program performs comparative analysis, coumarone-Vera index analysis, and sample uniformity analysis. The analysis results are shown in fig. 3 and 4.
FIG. 3 shows that the flora structures of bacteria in drainage and production water of coal-bed gas wells mostly belong to Proteobacteria and Bacteroides, and the flora structures of different samples are mostly different in the rest 4% of classifications, including Spiraria, Mycoplasma wartii and Gliocladium.
Fig. 4 shows that the species uniformity of the bacteria in the water sample is high, which is consistent with calculating the sample uniformity to be 0.77. In addition, the shannon-Vera index (H) is 2.15, which shows that the diversity of the bacteria in the water sample is not very high, and the Simpson index is 0.88, which also proves that the diversity of the bacteria in the sample is not high.
In the embodiment, drainage and production water of the coal-bed gas well is used as a bacteria source, a proper amount of culture medium is added under the laboratory condition, anthracite retrieved from a drainage and production well is used as a substrate, and the anthracite is cultured in an anaerobic bottle. Collecting culture solution on different nodes of a culture period, and rapidly detecting the change of the flora structure in drainage and production water of the coal-bed gas well by a PCR-DGGE method.
The flora structure in the coal bed water in the anaerobic reactor in an experimental period is changed continuously, the method can quickly and efficiently detect and know the dynamic change of the flora structure on each node in the experimental period, and the target sequence is analyzed in a targeted manner. The existing high-throughput sequencing technology has the advantages that the cycle for testing a sample is about 30 days, the whole sequence is sequenced, and the cost is higher. The detection period of the method is 3 days, preliminary judgment can be made by observing lane strip changes, if large changes are found, gel cutting and sequencing analysis are performed in a targeted manner, multiple times of high-throughput sequencing can be avoided, and the method can be fast, efficient and cost-saving.
Claims (5)
1. A method for rapidly detecting a flora structure in drainage and production water of a coal-bed gas well by utilizing PCR-DGGE is characterized by comprising the following steps: anaerobic collection of drainage and collection water samples is carried out on a drainage and collection site of the coal bed gas well, the site liquid nitrogen is stored and brought back to a laboratory for storage at 4 ℃, the obtained water samples are used as a bacteria source, then a culture medium is added, anthracite retrieved from a drainage and collection well is used as a substrate, and the anthracite is cultured in an anaerobic bottle; collecting culture solution at several nodes of a culture period of an anaerobic gas production experiment, extracting DNA, performing PCR amplification by using a bacterial 16S rDNA V1-V3 variable region design primer, and performing DGGE electrophoresis separation on fragments and recovering; and (5) sequencing after the second amplification, and detecting the change of the flora structure in the drainage and production water of the coal-bed gas well.
2. The method for rapidly detecting the flora structure in drainage and production water of the coal-bed gas well by using the PCR-DGGE as claimed in claim 1, wherein the method comprises the following steps: the method comprises the following specific steps:
(1) culturing strains: taking a water sample collected from a coal bed well which is preserved in an anaerobic way as a bacteria source, adding a culture medium and a coal sample, and culturing in an anaerobic bottle; taking a 500ml anaerobic bottle, adding 25g of coal powder as a substrate in an anaerobic box, adding 250ml of culture medium and 50ml of water sample, sealing, and culturing in a constant-temperature incubator at 35 ℃;
(2) and (3) extracting DNA: collecting bacterial liquid at a plurality of time nodes in a culture period, filtering and collecting thalli, and extracting DNA and storing at-20 ℃;
(3) sequence amplification and purification: performing PCR sequence amplification twice and purifying:
for the first amplification, a pair of universal primers is designed according to a variable region of 16S rDNA V1-V3 of bacteria for PCR amplification, wherein the sequence of an upstream primer is 27F: 5-AGAGT TTGAT CCTGG CTCAG-3;
the downstream primer is 518R: 5-ATTAC CGCGG CTGCT GG-3;
the PCR amplification condition is pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 65 ℃ for 1min, extension at 72 ℃ for 1min, and 25 cycles; extending for 10min at 72 ℃;
for the second amplification, a pair of universal primers is designed according to the variable region of the bacteria 16S rDNA V3 for PCR, wherein a hairpin framework of 30-40bp is inserted into the 5' end of the upstream primer; the PCR upstream primer sequence is 338F-GC: 5-CGCCC GCCGC GCGCGGCGGG CGGGG CGGGG GCACG GGGGG ACTCC TACGG GAGGC AGCAG-3;
the sequence of the downstream primer is 518R: 5-ATTAC CGCGG CTGCT GG-3;
wherein the sequence of the GC hairpin structure is 5-CGCCC GCCGC GCGCG GCGGG CGGGG CGGGG-3;
the PCR amplification conditions comprise pre-denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 1min, extension at 72 ℃ for 2min and 25 cycles; extending for 10min at 72 ℃;
detecting PCR amplification products by 2% gel electrophoresis;
(4) DGGE electrophoresis and imaging observations: the concentration of polyacrylamide gel is 10 percent, the gel denaturation gradient is 40 to 60 percent, and the Buffer solution is 1 multiplied by TAE Buffer; polymerizing the denatured gel for 90 minutes, then inserting a 16-comb into the space at the top of the denatured gel, adding 25 mu L of PCR product into each hole by using a sample injection needle, carrying out electrophoresis at the temperature of 60 ℃ and the voltage of 200V for 5min, and then carrying out electrophoresis at the temperature of 60 ℃ and the voltage of 85V for 12 h; then dyeing in 3 XGelRed dye for 25-35min, and observing gel imaging under an ultraviolet lamp of a gel irradiation instrument;
(5) cutting gel, recovering a target band, and sequencing: marking the clear common and specific bands in the DGGE map with cut gel, putting the cut gel into a 1.5mL centrifuge tube, adding 20 mu L deionized water, mashing the gel, dissolving the gel, standing overnight at 4 ℃, and sequencing a dissolving solution;
(6) analyzing a sequencing result: and performing comparison analysis, coumarone-Vera index analysis and sample uniformity analysis by using an NCBI Blast program to determine main variable strains of the bacterial liquid samples taken from a plurality of nodes.
3. The method for rapidly detecting the flora structure in drainage and production water of the coal-bed gas well by using the PCR-DGGE as claimed in claim 2, wherein the method comprises the following steps: the culture medium in the step (1) is an inorganic culture medium, and the formula of the culture medium is 2g/L K2HPO4、1.5g/LKH2PO4、0.4g/L MgCl2、1g/L NH4Cl, 2g/L YE; the coal sample is anthracite in a drainage and mining underground coal bed.
4. The method for rapidly detecting the flora structure in drainage and production water of the coal-bed gas well by using the PCR-DGGE as claimed in claim 2, wherein the method comprises the following steps: one culture period in the step (2) is 25-35 days; the plurality of time nodes are a gas production early stage, a gas production rising stage, a gas production stable stage and a gas production descending stage.
5. The method for rapidly detecting the flora structure in drainage and production water of the coal-bed gas well by using the PCR-DGGE as claimed in claim 2, wherein the method comprises the following steps: comparing and analyzing the sequencing result by using an NCBI Blast program in the step (6) to determine common and specific strains in the flora structures of different samples; and obtaining the number, brightness and position specific data of the strips by contrasting the DGGE map, and obtaining the change condition of the flora structure in different samples.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100704986B1 (en) * | 2006-04-27 | 2007-04-09 | 한국생명공학연구원 | Establishment of functional microbial community being capable of degrading diesel oil and the preservation method |
| CN102533964A (en) * | 2010-12-24 | 2012-07-04 | 云南天士力帝泊洱生物茶集团有限公司 | Predominant bacteria in microbial population of Pu'er tea and spectrum for predominant bacteria |
| CN103981259A (en) * | 2014-05-06 | 2014-08-13 | 山西晋城无烟煤矿业集团有限责任公司 | Analysis method for diversity of microbes and abundance of species in coal seam water |
| CN107099604A (en) * | 2017-06-02 | 2017-08-29 | 湖南省硕远检测技术有限公司 | Method for detecting microorganism in underground water of coal bed methane field |
-
2019
- 2019-12-25 CN CN201911355529.4A patent/CN111041118A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR100704986B1 (en) * | 2006-04-27 | 2007-04-09 | 한국생명공학연구원 | Establishment of functional microbial community being capable of degrading diesel oil and the preservation method |
| CN102533964A (en) * | 2010-12-24 | 2012-07-04 | 云南天士力帝泊洱生物茶集团有限公司 | Predominant bacteria in microbial population of Pu'er tea and spectrum for predominant bacteria |
| CN103981259A (en) * | 2014-05-06 | 2014-08-13 | 山西晋城无烟煤矿业集团有限责任公司 | Analysis method for diversity of microbes and abundance of species in coal seam water |
| CN107099604A (en) * | 2017-06-02 | 2017-08-29 | 湖南省硕远检测技术有限公司 | Method for detecting microorganism in underground water of coal bed methane field |
Non-Patent Citations (2)
| Title |
|---|
| KAMILA ORLEWSKA等: "Use of the PCR-DGGE Method for the Analysis of the Bacterial Community Structure in Soil Treated With the Cephalosporin Antibiotic Cefuroxime and/or Inoculated With a Multidrug-Resistant Pseudomonas putida Strain MC1", 《FRONTIERS IN MICROBIOLOGY》 * |
| 赵晗等: "内蒙胜利褐煤生物产气前后微生物群落变化", 《煤炭学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112626243A (en) * | 2020-12-24 | 2021-04-09 | 山西大学 | Kit and method for rapidly detecting bacteria in coal geological environment |
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