CN112646902A

CN112646902A - Kit and method for rapidly detecting archaea

Info

Publication number: CN112646902A
Application number: CN202011553226.6A
Authority: CN
Inventors: 杨秀清; 徐现
Original assignee: Shanxi University
Current assignee: Shanxi University
Priority date: 2020-12-24
Filing date: 2020-12-24
Publication date: 2021-04-13

Abstract

The invention belongs to the field of microbial molecular ecology detection, in particular to a kit and method for rapid detection of archaea. Aiming at the problems existing in real-time quantitative PCR and high-throughput sequencing to explore the abundance, diversity, composition and structure of archaea communities in different environments, the present invention is a kit for rapid archaea detection, including primer sets and DNA fragments, PCR buffer and STE buffer. In addition, the invention utilizes the DGGE technology to rapidly detect archaea, has the advantages of low experimental cost, simple operation, strong specificity, easy popularization in the laboratory, and no dependence on large-scale experimental equipment. It is an efficient, fast, economical and convenient detection method.

Description

Kit and method for rapidly detecting archaea

Technical Field

The invention belongs to the field of microbial molecular ecology detection, relates to a gel electrophoresis (DGGE) technology related to environmental microbial community detection, and particularly relates to a kit and a method for rapidly detecting archaea.

Background

Archaea is the third form of life, and contains information in the early stage of life evolution. The archaea is widely distributed in various environments (including extreme natural environments) on the earth, is extremely rich in genetic and metabolic diversity and is an important driver of the geochemical cycle of element biology. Meanwhile, the archaea and metabolites thereof also exhibit unique biotechnological development potential.

Methanogenic bacteria are a very special biological group, belonging to the archaea. The bacteria have special energy-producing metabolism function, and can utilize organic or inorganic substances as substrates to convert into methane under anaerobic conditions. The methanogenic bacteria also have important significance in the dynamic balance of carbon substance circulation in nature. Methanogenic bacteria can live in various anaerobic environments, even some extreme environments, and these adaptations are a result of long-term evolution and exist in the treatment of organic waste, organic matter of biogas fermentation, swamps, lakes, marine sediments, and the treatment of high-concentration organic wastewater, among others. Meanwhile, the archaea has abundant metabolic diversity. Therefore, archaea is considered to play an important role in the biogeochemical cycle of driver elements. On the other hand, since most of the microorganisms are extreme microorganisms, archaea and metabolites thereof have potential biotechnological development values.

At present, the abundance, diversity, composition and structure of archaea communities in different environments are mostly explored by adopting real-time fluorescent quantitative PCR and high-throughput sequencing, and the archaea is detected by the real-time fluorescent quantitative PCR under the following conditions: the price of the required reagent is high, the use times of the experiment are few, and the mass detection and identification of the archaea cannot be carried out. Although the gene sequencing is simple and convenient to operate, the problems of high sequencing cost and high sequencing error rate exist, a large scientific research instrument and a professional biological information analyzer are relied on, and the detection period is long.

Disclosure of Invention

Aiming at the problems, the invention provides a kit and a method for rapidly detecting archaea.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides a primer group and a DNA fragment for rapid detection of archaea, wherein the sequences of the primer group members are shown as SEQ ID NO: 1. SEQ ID NO: 2. seq id no: 3 is shown in the specification; the DNA fragment has a sequence shown in SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10. SEQ ID NO: shown at 11.

Further, the primer set and the DNA fragment are nucleotide fragments synthesized by chemical synthesis.

The invention provides a kit for rapid detection of archaea, which comprises: the primer group, DNA fragment, PCR buffer solution and STE buffer solution.

Further, the PCR Buffer included 5. mu.l of 10 × Easy Taq Buffer, 4. mu.l of 10mmol/L dNTPs, 1. mu.l of the upstream primer, 1. mu.l of the downstream primer, 1. mu.l of Taq enzyme, 1.5. mu.l of the microorganism genomic DNA, 36.5. mu.l of dd H₂O。

The STE buffer solution was prepared by dissolving 0.584g of NaCl, 0.12g of Tris and 0.37g of EDTA in 100ml of double distilled water.

The invention provides a method for rapidly detecting archaea, which comprises the following steps:

step 1, extracting total DNA of a microbial genome of a sample;

step 2, respectively using ultrapure water, a chemically synthesized DNA fragment and a sample microbial genome total DNA as templates to carry out PCR amplification, wherein an ultrapure water amplification product is used as a negative control of a sample to be detected, and a DNA fragment amplification product is used as a standard of the sample to be detected; the PCR amplification product of the sample is used as a sample to be detected for DGGE analysis and detection;

step 3, carrying out DGGE detection on the standard sample and the sample to be detected;

step 4, after the DGGE detection is finished, putting the gel containing the chemically synthesized DNA fragments and the sample to be detected into 2.5 multiplied by 4S Red Plus dye solution for dyeing for 15 minutes, photographing for analysis and comparison, and when a certain strip of the sample to be detected and a standard strip are positioned at the same horizontal position on the DGGE gel, indicating that the sample to be detected contains the archaea shown by the standard strip; and when the sample to be detected has no strip or the strip and the standard strip are not in the same horizontal position on the DGGE gel, indicating that the sample to be detected has no corresponding archaea.

Further, the step of extracting total DNA of the microbial genome of the sample in the step 1 is as follows:

(1) soaking the sample in STE buffer solution, filtering the sample with 0.22 μm filter membrane, and storing the filter membrane for retaining microorganism at-20 deg.C;

(2) washing the filter membrane with STE buffer solution to obtain filtrate, and centrifuging at 12000rpm for 1 min;

(3) adding 300 mu l of STE buffer solution and 30 mu l of lysozyme into the precipitate, flushing the mixture by using a pipette, uniformly mixing the mixture, and carrying out thermostatic water bath for 3.5h at 37 ℃;

(4) continuously adding 35 μ l of SDS and 5 μ l of protease K, carrying out thermostatic water bath for 2.5h at 55 ℃, taking out after completion, cooling to room temperature, and adding 70 μ l of NaCl;

(5) adding 450 μ l DNA phenol extraction reagent, mixing, centrifuging at 12000rpm for 10min, and collecting supernatant;

(6) adding the mixture in an equal volume ratio of 25: 24: 1, extracting with phenol-chloroform-isoamyl alcohol, centrifuging at 12000rpm for 10min, taking supernatant, and repeating once;

(7) adding 2 times of isopropanol to precipitate DNA, standing, removing supernatant, and collecting precipitate;

(8) washing the precipitate with 75% ethanol solution, purging, standing for 2 min, and sucking out ethanol;

(9) add 40. mu.l ddH2O and 2. mu.l RNase, bath at 55 ℃ for 30min, and store in a 4 ℃ incubator overnight for further use.

The DEGG detection in the step 3 is as follows: 8 percent of polyacrylamide gel is selected, the gel modification range is 40 to 60 percent, and the polyacrylamide gel is mixed in a gradient way to prepare gel; when the temperature of the electrophoretic liquid rises to 60 ℃, performing 220V constant-pressure pre-electrophoresis for 5min, and quickly loading the sample by using a 50 mu L microsyringe, wherein the loading amount is 30 mu L; performing electrophoresis at constant temperature and constant voltage for 8h at 60 ℃ and 85V.

Compared with the prior art, the invention has the following advantages:

the invention utilizes DGGE technology, has the advantages of low experimental cost, simple and convenient operation, strong specificity, easy popularization in laboratories, no dependence on large-scale experimental equipment and the like. The method is an efficient, rapid, economic and convenient detection method.

Drawings

FIG. 1 is a graph showing the results of detection in example 1.

FIG. 2 is a graph showing the results of detection in example 2.

FIG. 3 is a graph showing the results of detection in example 3.

In the figure, M represents a chemically synthesized DNA standard molecular fragment of archaea.

Detailed Description

The technical solution in the embodiments of the present invention will be specifically and specifically described below with reference to the embodiments of the present invention and the accompanying drawings. It should be noted that variations and modifications can be made by those skilled in the art without departing from the principle of the present invention, and these should also be construed as falling within the scope of the present invention.

Example 1

1. Chemically synthesizing primer group and DNA segment for fast detection of archaea.

Wherein the primer group member sequence is SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3; all DNA fragment sequences are SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8. SEQ ID NO: 9. SEQ ID NO: 10, or a fragment thereof.

2. The chemically synthesized DNA fragments were each amplified using a primer set of SEQ ID NO: 1 (upstream primer)/SEQ ID NO: 3 (downstream primer) to carry out PCR amplification, and taking an amplification product as a standard of a sample to be detected.

The PCR reaction system is as follows: 10 easyltaq Buffer: 5 μ L, dNTPs (2,5 mM): 4 μ L, forward primer (10 μmol/L): 1 μ L, downstream primer (10 μmol/L): 1 μ L, EasyTaq DNA Polymerase: 1 μ L, template DNA (chemically synthesized DNA fragment): 1 μ L, ddH 20: 37 mu L of the solution;

the PCR reaction program is: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 45-55 ℃ for 30s and 72 ℃ for 30 s; preserving at 72 deg.C for 10min and 12 deg.C.

3. The DNA fragments used as standards were subjected to DGGE detection and validation:

selecting 8% polyacrylamide gel with a gel modification range of 40% -60%, taking 16ml of each of 40% gel and 60% gel, respectively adding 50 muL TEMED and 40 muL 10% ammonium persulfate, and mixing and preparing the gel in a gradient manner for at least 3 h; before sample adding, when the temperature of the electrophoresis liquid is raised to 55 ℃, carrying out 220V pre-electrophoresis for 5min, wherein the sample loading amount of a PCR amplification product is 30 mu L, and carrying out electrophoresis for 8h under the conditions of 55 ℃ and 85V; after electrophoresis, the gel was stained in 2.5X 4S Red Plus dye for 15 minutes, photographed for observation, and analyzed as shown in FIG. 1.

Example 2

The method for detecting activated sludge archaea in an aeration tank of a certain sewage treatment plant comprises the following steps: the method comprises the following steps:

1. extracting total DNA of activated sludge microbial genome in aeration tank

(1) The activated sludge was diluted and dissolved with ultrapure water, suction-filtered with a 0.22 μm filter, and the filter with the microorganisms was placed in an EP tube, washed twice with STE buffer, and centrifuged at 12000rpm for 1 minute.

(2) Adding STE buffer solution 300. mu.l and lysozyme 30. mu.l, flushing with a pipette, mixing, and thermostatic water bath at 37 ℃ for 3.5 hours.

(3) SDS 35. mu.l and Proteinase K5. mu.l were added thereto, and the mixture was incubated in a thermostatic water bath at 55 ℃ for 2.5 hours.

(4) After completion, the reaction mixture was taken out, cooled to room temperature, and 70. mu.l of NaCl was added.

(5) Add 450. mu.l DNA extraction phenol reagent, mix well for 10 minutes, centrifuge at 12000rpm for 10 minutes, take the supernatant.

(6) Adding phenol-chloroform-isoamyl alcohol (volume ratio is 25: 24: 1) with the same volume, extracting, centrifuging at 12000rpm for 10 minutes, and taking supernatant. And repeating the steps once.

(7) Adding 2 times of isopropanol to precipitate DNA, standing, removing supernatant, and collecting precipitate.

(8) The precipitate was washed twice with 75% ethanol solution, purged, left to stand for 2 minutes and the ethanol aspirated.

(9) Mu.l of ddH2O and 2. mu.l of RNase were added and the mixture was washed with water at 55 ℃ for 30 minutes.

(10) And (4) putting the substance obtained in the step (9) into a constant temperature box at 4 ℃ for storage overnight, performing electrophoresis by using 1.5% agarose gel, and detecting the size and integrity of the extracted genome DNA for later use.

2. Respectively carrying out PCR amplification on ultrapure water, chemically synthesized DNA fragments and aeration tank activated sludge microorganism total genome DNA

2.1 Primary amplification of Total genome fragments to be detected extracted from activated sludge in aeration tank

The total genome DNA of the microorganism in the activated sludge of the aeration tank obtained in the previous step is taken as a template, and a primer pair SEQ ID NO: 1/SEQ ID NO: 2, carrying out PCR amplification on 16S rRNA fragments of different archaea. The PCR reaction system is a 50-microliter reaction system, and the reaction conditions refer to one-time PCR amplification of the gene fragment of the 16S rRNA of the archaea in the environment.

2.2 detection and recovery of PCR amplification products

Detecting the PCR product obtained by the primary amplification by using 1.5% agarose gel electrophoresis, cutting a corresponding band of the detection gel of the amplification product under the irradiation of an ultraviolet lamp, and recovering the PCR product according to a gel recovery kit.

2.3 Secondary amplification of Total genome fragments to be detected in the activated sludge extracted from the aeration tank

Taking the gel recovered product as a template, and using a primer pair SEQ ID NO: 3/SEQ ID NO: 2 Secondary amplification of the 16SrRNA gene is performed, and the PCR amplification system and the reaction conditions refer to the conditions of the primary PCR amplification. Meanwhile, ultrapure water and chemically synthesized DNA fragments are respectively used as templates for PCR amplification and respectively used as a negative control and a standard.

DGGE analysis

Selecting 8% polyacrylamide gel with gel denaturation range of 40% -60%, taking 15ml of 40% and 60% gel, respectively adding 50 μ l TEMED and 40 μ l 10% APS, and gradient mixing to obtain gel. After filling, the comb was gently inserted and allowed to solidify at room temperature for 3 hours. After complete solidification, the comb is pulled out, the whole plate is arranged on a DGGE support, the DGGE support provided with the plate is placed in an electrophoresis tank, when the temperature of the electrophoresis liquid rises to 55 ℃, the electrophoresis is performed for 5min under the constant pressure of 220V, a 50 mu l microsyringe is used for rapid sample loading, the sample loading is ultrapure water, a chemically synthesized DNA fragment and a secondary PCR amplification product of activated sludge in an aeration tank, and the sample loading amount is 30 mu l. Electrophoresis was carried out at constant temperature and constant pressure at 55 ℃ and 85V for 8 hours. After the electrophoresis was completed, the gel was stained in 2.5 × 4S Red Plus dye for 15 minutes, and then placed in an ultraviolet analyzer for photographing with a purple tray, and as a result, it was found from fig. 2 that the activated sludge in the aeration basin in this sewage treatment plant was rich in four types of archaea, methanopirillum, methanoarcina and methanopirillum.

Example 3

The method for detecting the archaea in the sediments of the rural biogas digester comprises the following steps: the method comprises the following steps:

1. extracting total DNA of microbial genome in sediment of methane tank

1.1 DNA extraction

(1) Diluting the solid-liquid sample in the methane tank with ultrapure water, performing suction filtration with a 0.22 μm filter membrane, placing the filter membrane with the archaea microorganisms into an EP tube, washing twice with STE buffer solution, and centrifuging at 12000rpm for 1 minute.

(9) Add 40. mu.l of ddH₂O, 2. mu.l of RNase, and water bath at 55 ℃ for 30 minutes.

2. Respectively carrying out PCR amplification on ultrapure water, chemically synthesized DNA fragments and archaea microorganism total genome DNA of the methane tank

2.1 Primary amplification of the fragments to be detected of the Total genome of the microorganisms of the sediments of biogas digesters

The microbial total genome DNA obtained in the previous step is taken as a template, and a primer pair SEQ ID NO: 1/SEQ ID NO: 2, carrying out PCR amplification on 16S rRNA fragments of different archaea. The PCR reaction system is a 50-microliter reaction system, and the reaction conditions refer to one-time PCR amplification of the gene fragment of the 16S rRNA of the archaea in the environment.

2.2 detection and recovery of PCR amplification products

2.3 Secondary amplification of Total genomic fragments to be detected extracted from biogas digester precipitate microorganisms

Taking the gel recovered product as a template, and using a primer pair SEQ ID NO: 3/SEQ ID NO: 2 carrying out secondary amplification of different archaea 16SrRNA genes, wherein the PCR amplification system and the reaction conditions refer to the conditions of the primary PCR amplification. Meanwhile, ultrapure water and chemically synthesized DNA fragments are respectively used as templates for PCR amplification and respectively used as a negative control and a standard.

DGGE analysis

Selecting 8% polyacrylamide gel with gel denaturation range of 40% -60%, taking 15ml of 40% and 60% gel, respectively adding 50 μ l TEMED and 40 μ l 10% APS, and gradient mixing to obtain gel. After filling, the comb was gently inserted and allowed to solidify at room temperature for 3 hours. After complete solidification, the comb is pulled out, the whole plate is arranged on a DGGE bracket, the DGGE bracket provided with the plate is placed in an electrophoresis tank, when the temperature of the electrophoresis liquid rises to 55 ℃, the electrophoresis is performed for 5min under the constant pressure of 220V, a 50 mu l microsyringe is used for rapid sample loading, the sample loading is ultrapure water, chemically synthesized DNA fragments and secondary PCR amplification products of archaea microorganisms of the methane tank, and the sample loading amount is 30 mu l. Electrophoresis was carried out at constant temperature and constant pressure at 55 ℃ and 85V for 8 hours. After electrophoresis, the gel was stained in 2.5X 4S Red Plus stain for 15 minutes and photographed with a purple tray in a UV analyzer (see FIG. 3). From the results, it was found that the biogas digester precipitate detection is rich in four types of archaea including Methanomorphucuum, Methanosarcina, Methanospirillum and Methanosarcina.

Sequence listing

SEQ ID NO：1

ccctacggggcgcagcag

SEQ ID NO：2

ggattacaagatttcac

SEQ ID NO：3

cgcccgccgcgcgcggcgggcggggcgggggcacggggggggattacaagatttcac

SEQ ID NO：4

ccctacggggcgcagcaggcgcgcaaactttacaatgcgagcaatcgtgataaggaaaccctgagtgcctgtcgatgcaggctgttcatatatctaaatcatatgtgaagaaagggcagggcaagaccggtgccagccgccgcggtaataccggctgctcgagtgatggccactattactgggtttaaagcgtccgtagcttgactgttaggtctcttgggaaatcttcacgctcaacgtgaaggcgtctaagagataccggcagtcttggaactgggagaggtaaaccgtacttcgggggtaggagtgaaatcttgtaatcc

SEQ ID NO：5

SEQ ID NO：6

ccctacggggcgcagcaggcgcgaaaactttacaatgcgggaaaccgtgataaggggacaccgagtgccagcatcatatgctggctgtccgggtgtgtaaaatacacctgttagcaagggccgggcaagaccggtgccagccgccgcggtaacaccggcggcccgagtggtgatcgtgattattgggtctaaagggtccgtagccggtttggtcagtcctccgggaaatctgatagctcaactattaggctttcgggggatactgccagacttggaaccgggagaggtaagaggtactacaggggtaggagtgaaatcttgtaatcc

SEQ ID NO：7

ccctacggggcgcagcaggcgcgaaaactttaccatgcgggcaaccgtgataaggaaaccccgagtgccagcacaggctggctgtccaccagtgtaaataactggtgaagaaagggccgggcaagaccggtgccagccgccgcggtaataccggcggctcgagtggtggccgctattactgggcttaaagggtccgtagctggatatacaagtcccttgagaaatccgccggcttaaccggtgggcgttcaggggaaactgtatttctagggaccgggagaggtgagaggtactgccggggtaggagtgaaatcctgtaatcc

SEQ ID NO：8

SEQ ID NO：9

gcctacggggcgcagcaggcgcgaaaactttaacatgcgggcaaccgtgataaggaaaccccgagtgccagcacaggctggctgtccaccagtgtaaataactggtgaagaaagggccgggcaagaccggtgccagccgccgcggtaataccggcggctcgagtggtggccactattactgggcttaaagggtccgtagcttgatatgcaagtcttttgagaaatccgccggcttaactggtgggcgttcaggggaaactgcatttctagggaccgggagaggtgagaggtactgccggggtaggagtgaaatcctgtaatcc

SEQ ID NO：10

SEQ ID NO：11

ccctacggggtgcagcaggcgcgaaacctttacaatacgggaaaccgtgataagggaatctcgagtgccagcatacaatgttggctgtccagatgcctaaaaagcatctgttagcaagggccgggcaagaccggtgccagccgccgcggtaacaccggcggcccgagtggtaaccgctattattgggtctaaagggtctgtagccggccaagtaagtcccttgggaaatctggcagcttaactgtcaggctgccaggggatactgtttggcttgggaccgggagaggtgagaggtacctcaagggtaggggtgaaatcttgtgatcc

Sequence listing

<110> university of Shanxi

<120> kit and method for rapid detection of archaea

<160> 11

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ccctacgggg cgcagcag 18

<210> 2

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggattacaag atttcac 17

<210> 3

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgcccgccgc gcgcggcggg cggggcgggg gcacgggggg ggattacaag atttcac 57

<210> 4

<211> 323

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ccctacgggg cgcagcaggc gcgcaaactt tacaatgcga gcaatcgtga taaggaaacc 60

ctgagtgcct gtcgatgcag gctgttcata tatctaaatc atatgtgaag aaagggcagg 120

gcaagaccgg tgccagccgc cgcggtaata ccggctgctc gagtgatggc cactattact 180

gggtttaaag cgtccgtagc ttgactgtta ggtctcttgg gaaatcttca cgctcaacgt 240

gaaggcgtct aagagatacc ggcagtcttg gaactgggag aggtaaaccg tacttcgggg 300

gtaggagtga aatcttgtaa tcc 323

<210> 5

<211> 323

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ccctacgggg cgcagcaggc gcgcaaactt tacaatgcga gcaatcgtga taaggaaacc 60

ctgagtgcct gtcgatgcag gctgttcata tatctaaatc atatgtgaag aaagggcagg 120

gcaagaccgg tgccagccgc cgcggtaata ccggctgctc gagtgatggc cactattact 180

gggtttaaag cgtccgtagc ttgactgtta ggtctcttgg gaaatcttca cgctcaacgt 240

gaaggcgtct aagagatacc ggcagtcttg gaactgggag aggtaaaccg tacttcgggg 300

gtaggagtga aatcttgtaa tcc 323

<210> 6

<211> 327

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ccctacgggg cgcagcaggc gcgaaaactt tacaatgcgg gaaaccgtga taaggggaca 60

ccgagtgcca gcatcatatg ctggctgtcc gggtgtgtaa aatacacctg ttagcaaggg 120

ccgggcaaga ccggtgccag ccgccgcggt aacaccggcg gcccgagtgg tgatcgtgat 180

tattgggtct aaagggtccg tagccggttt ggtcagtcct ccgggaaatc tgatagctca 240

actattaggc tttcggggga tactgccaga cttggaaccg ggagaggtaa gaggtactac 300

aggggtagga gtgaaatctt gtaatcc 327

<210> 7

<211> 323

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ccctacgggg cgcagcaggc gcgaaaactt taccatgcgg gcaaccgtga taaggaaacc 60

ccgagtgcca gcacaggctg gctgtccacc agtgtaaata actggtgaag aaagggccgg 120

gcaagaccgg tgccagccgc cgcggtaata ccggcggctc gagtggtggc cgctattact 180

gggcttaaag ggtccgtagc tggatataca agtcccttga gaaatccgcc ggcttaaccg 240

gtgggcgttc aggggaaact gtatttctag ggaccgggag aggtgagagg tactgccggg 300

gtaggagtga aatcctgtaa tcc 323

<210> 8

<211> 327

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ccctacgggg cgcagcaggc gcgaaaactt tacaatgcgg gaaaccgtga taaggggaca 60

ccgagtgcca gcatcatatg ctggctgtcc gggtgtgtaa aatacacctg ttagcaaggg 120

ccgggcaaga ccggtgccag ccgccgcggt aacaccggcg gcccgagtgg tgatcgtgat 180

tattgggtct aaagggtccg tagccggttt ggtcagtcct ccgggaaatc tgatagctca 240

actattaggc tttcggggga tactgccaga cttggaaccg ggagaggtaa gaggtactac 300

aggggtagga gtgaaatctt gtaatcc 327

<210> 9

<211> 323

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gcctacgggg cgcagcaggc gcgaaaactt taacatgcgg gcaaccgtga taaggaaacc 60

ccgagtgcca gcacaggctg gctgtccacc agtgtaaata actggtgaag aaagggccgg 120

gcaagaccgg tgccagccgc cgcggtaata ccggcggctc gagtggtggc cactattact 180

gggcttaaag ggtccgtagc ttgatatgca agtcttttga gaaatccgcc ggcttaactg 240

gtgggcgttc aggggaaact gcatttctag ggaccgggag aggtgagagg tactgccggg 300

gtaggagtga aatcctgtaa tcc 323

<210> 10

<211> 323

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gcctacgggg cgcagcaggc gcgaaaactt taacatgcgg gcaaccgtga taaggaaacc 60

ccgagtgcca gcacaggctg gctgtccacc agtgtaaata actggtgaag aaagggccgg 120

gcaagaccgg tgccagccgc cgcggtaata ccggcggctc gagtggtggc cactattact 180

gggcttaaag ggtccgtagc ttgatatgca agtcttttga gaaatccgcc ggcttaactg 240

gtgggcgttc aggggaaact gcatttctag ggaccgggag aggtgagagg tactgccggg 300

gtaggagtga aatcctgtaa tcc 323

<210> 11

<211> 327

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ccctacgggg tgcagcaggc gcgaaacctt tacaatacgg gaaaccgtga taagggaatc 60

tcgagtgcca gcatacaatg ttggctgtcc agatgcctaa aaagcatctg ttagcaaggg 120

ccgggcaaga ccggtgccag ccgccgcggt aacaccggcg gcccgagtgg taaccgctat 180

tattgggtct aaagggtctg tagccggcca agtaagtccc ttgggaaatc tggcagctta 240

actgtcaggc tgccagggga tactgtttgg cttgggaccg ggagaggtga gaggtacctc 300

aagggtaggg gtgaaatctt gtgatcc 327

Claims

1. a primer set and a DNA fragment for the rapid detection of archaea, characterized in that the primer set comprises sequences such as those shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 Primers; the DNA fragments include fragments whose sequences are shown in SEQ ID NOs: 4-11.

2 . The primer set and DNA fragment for rapid archaea detection according to claim 1 , wherein the primer set and the DNA fragment are chemically synthesized nucleotide fragments. 3 .

3. The kit for rapid detection of archaea according to claim 1, characterized in that, comprising: the primer set and DNA fragment according to claim 1, a PCR buffer, and a STE buffer.

4. The kit for rapid detection of archaea according to claim 3, wherein the PCR buffer comprises 5 μl 10×Easy Taq Buffer, 4 μl 10mmol/L dNTPs, 1 μl upstream primer, 1 μl downstream primer Primers, 1 μl Taq enzyme, 1.5 μl DNA fragment, 36.5 μl ddH2O.

5. a kind of kit for archaea rapid detection according to claim 3, is characterized in that: described STE buffer solution is dissolved in 100ml by the NaCl of 0.584g, the Tris of 0.12g and the EDTA of 0.37g Made with double distilled water.

6. a method for the rapid detection of archaea described in any one of claim 1-5, is characterized in that, comprises the following steps:

Step 1, extract the total DNA of the sample microbial genome;

Step 2: PCR amplification is carried out with ultrapure water, chemically synthesized DNA fragments and the total DNA of the sample microbial genome as templates respectively, the amplification product of ultrapure water is used as the negative control of the sample to be tested, and the amplified product of the DNA fragment is used as the sample to be tested standard; the PCR amplification product of the sample is used as the sample to be tested for DGGE detection;

Step 3, carry out DGGE detection on the standard and the sample to be tested;

Step 4. After the DGGE detection is completed, put the gel containing the chemically synthesized DNA fragments and the sample to be tested in 2.5×4SRed Plus dye solution for 15 minutes, and take pictures for analysis and comparison. When the bands are in the same horizontal position on the DGGE gel, it means that the sample to be tested contains the archaea shown in the standard band; when the sample to be tested has no band or the band and the standard band are not in the same horizontal position on the DGGE gel , indicating that the test sample has no corresponding archaea.

7. the method for the rapid detection of a kind of archaea according to claim 6, is characterized in that, the step of sample microorganism genome total DNA extraction in described step 1 is as follows:

(1) After soaking the sample with STE buffer, filter the sample with a 0.22 μm filter membrane, and then store the filter membrane that retains the microorganisms at -20°C;

(2) Wash the filter membrane with STE buffer to obtain a filtrate, and then centrifuge at 12000 rpm for 1 min;

(3) Add 300 μl of STE buffer and 30 μl of lysozyme to the precipitation, rinse and mix with a pipette, and place in a constant temperature water bath for 3.5 hours at 37°C;

(4) Continue to add 35 μl of SDS and 5 μl of Proteinase K, keep in a constant temperature water bath at 55°C for 2.5 hours, take out after completion, cool to room temperature, and add 70 μl of NaCl;

(5) Add 450 μl of DNA extraction phenol reagent, mix well, centrifuge at 12,000 rpm for 10 min, and take the supernatant;

(6) adding an equal volume of phenol-chloroform-isoamyl alcohol with a volume ratio of 25:24:1 for extraction, centrifuging at 12000 rpm for 10 min, taking the supernatant, and repeating once;

(7) adding 2 times the volume of isopropanol to precipitate the DNA, discarding the supernatant after standing, and taking the precipitate;

(8) Wash the precipitate with 75% ethanol solution, rinse, and suck out the ethanol after standing for 2 minutes;

(9) Add 40 μl ddH ₂ O and 2 μl RNase, place in a water bath at 55° C. for 30 min, and store in a 4° C. incubator overnight until use.

8 . The method for rapid detection of archaea according to claim 6 , wherein in the step 3, DEGG detection is as follows: 8% polyacrylamide gel is selected, and the gelatinization range is 40%-60%. 9 . , gradient mixing gel preparation; when the temperature of the electrophoresis solution rises to 55 ℃, pre-electrophoresis at 220V constant voltage for 5 min, and quickly load the sample with a 50 μL micro-injector, the loading volume is 30 μl; under the condition of 55 ℃, 85V constant temperature and constant voltage electrophoresis 8h.