Degenerate primers for rapid screening and identification of microcystin-degrading bacteria and their application

Abstract

The present invention discloses degenerate primers for rapid screening and identification of microcystin-degrading bacteria and their applications. The present invention specifically discloses a degenerate primer pair consisting of primer mlrA-354F and primer mlrA-785R, and a method for rapid screening and identification of microcystin-degrading bacteria. The present invention collects all published mlrA gene sequences from the NCBI database, uses BioEdit software to perform multiple sequence alignment, designs degenerate primer pairs according to the conserved regions of the gene, performs PCR amplification and sequencing using the DNA of the strain to be tested as a template, compares the sequencing results with known sequences, and tests the MC degradation ability of strains with positive results to verify the effectiveness of the PCR screening method. All the positive strains obtained by the present invention can effectively degrade MC, indicating that the identification method of the present invention has a high accuracy rate, and can effectively shorten the screening cycle of degrading bacteria, greatly saving screening costs.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and relates to degenerate primers for rapid screening and identification of microcystin degrading bacteria and applications thereof, and specifically relates to the design of degenerate primers, and a method and a kit for rapid screening and identification of microcystin degrading bacteria using the degenerate primers. Background Art

[0002] With the increasing degree of eutrophication of water bodies, cyanobacteria blooms frequently occur in aquatic ecosystems around the world. The release of secondary metabolites - algae toxins caused by this has become a public environmental problem that seriously threatens the ecological environment and human health. Most algae toxins are water-soluble and directly pollute water bodies. Among them, microcystins (MC) are the most detected and most harmful algae toxins in cyanobacteria bloom pollution. MC can specifically bind to protein phosphatases in cells (especially liver and kidney cells) and strongly inhibit their activity, so it has a wide range of toxic effects and is a strong liver tumor promoter. MC has a ring structure and an interval double bond, which is quite stable in water bodies. How to effectively remove MC pollution is a difficult problem in the field of environmental science. Studies have confirmed that microbial degradation is one of the main ways to remove MC from polluted water bodies. Microorganisms come from the environment, and using the metabolism of microorganisms themselves to remove MC from water bodies is undoubtedly a safe, efficient and environmentally friendly pollution control method.

[0003] The screening of MC-degrading strains is an important part in the development of microbial remediation technology. At present, the screening of MC-degrading bacteria generally adopts the following method: the strain to be tested is inoculated in a liquid culture medium with MC as the only carbon source, and a shake flask experiment is carried out. The degradation rate is calculated by regularly measuring the content of the remaining MC in the culture medium, thereby determining the degradation ability of the strain. In general, the successful screening of MC-degrading bacteria requires the detection of a large number of strains to be tested, and the workload required to determine their degradation rates one by one is large. In addition, the conventional detection method relies on expensive MC pure products (commercially available MC pure products cost thousands of yuan per milligram) as degradation substrates, and the quantitative detection of MC requires the use of liquid chromatography technology, resulting in high detection costs and long detection cycles for MC. Therefore, it is urgent to develop a new MC-degrading bacteria screening method to solve the drawbacks revealed by traditional screening methods. It is very necessary to establish a fast, accurate, sensitive, efficient, convenient and low-cost MC-degrading strain screening method. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to screen and / or identify microcystin-degrading bacteria quickly, efficiently and / or at low cost.

[0005] In order to solve the above technical problems, the present invention first provides a degenerate primer pair for identifying or assisting in identifying microcystin-degrading bacteria, wherein the degenerate primer pair consists of primer mlrA-354F and primer mlrA-785R; the primer mlrA-354F is a single-stranded DNA molecule shown in SEQ ID No.1; the primer mlrA-785R is a single-stranded DNA molecule shown in SEQ ID No.2.

[0006] Furthermore, in the nucleotide sequence shown in SEQ ID No.1 and / or SEQ ID No.2, Y=C or T, V=G, A or C, S=C or G.

[0007] The primer mlrA-354F is a forward primer, and the primer mlrA-785R is a reverse primer.

[0008] The present invention also provides a reagent or a kit for identifying or assisting in identifying microcystin-degrading bacteria, wherein the reagent or the kit comprises the degenerate primer pair.

[0009] Furthermore, the kit also contains DNA lysis solution and PCR reaction solution.

[0010] The DNA lysis solution may include: Tris-HCl, EDTA, NaCl, and SDS.

[0011] The PCR reaction solution includes: upstream primer mlrA-354F, downstream primer mlrA-785R, and Premix Taq.

[0012] Premix Taq includes: DNA Polymerase, Buffer, dNTP.

[0013] The present invention also provides a method for identifying or assisting in identifying microcystin-degrading bacteria, the method comprising the following steps:

[0014] A1) extracting DNA from the sample to be tested;

[0015] A2) using the DNA of the sample to be tested as a template and the degenerate primer pair to perform PCR amplification; determining whether the sample to be tested contains microcystin-degrading bacteria or whether the sample to be tested is microcystin-degrading bacteria according to whether the PCR amplification product contains a DNA fragment of 449 bp in size.

[0016] In the above method, the method for determining whether the sample to be tested contains microcystin-degrading bacteria or whether the sample to be tested is microcystin-degrading bacteria according to whether the PCR amplification product contains a DNA fragment of 449 bp in size is: if the PCR amplification product contains a DNA fragment of 449 bp in size, then the sample to be tested contains microcystin-degrading bacteria or the sample to be tested is microcystin-degrading bacteria; if the PCR amplification product does not contain a DNA fragment of 449 bp in size, then the sample to be tested does not contain microcystin-degrading bacteria or the sample to be tested is not microcystin-degrading bacteria.

[0017] In the above method, the PCR amplification reaction procedure is: pre-denaturation at 95°C for 5 minutes; denaturation at 98°C for 10 seconds, annealing at 50°C for 30 seconds, extension at 72°C for 1 minute; 30 cycles; and further extension at 72°C for 10 minutes.

[0018] In the above method, the PCR amplification reaction system is: 2 μL of DNA lysis supernatant, 0.2 μM each of upstream primer mlrA-354F and downstream primer mlrA-785R, 25 μL of Premix Taq, and sterile distilled water to 50 μL.

[0019] In the above method, the method for extracting DNA from the sample to be tested includes: placing the sample to be tested in a lysis solution at 80° C. for 15 minutes.

[0020] In the above method, 1% agarose gel (EB staining) electrophoresis was used to detect the PCR amplification product.

[0021] The present invention also provides a method for screening and identifying microcystin-degrading bacteria using the degenerate primer pair, comprising the following steps: (1) collecting all published mlrA gene sequences from the NCBI database; (2) using BioEdit software to perform multiple sequence alignment and designing degenerate primer pairs based on the conserved regions of the gene; (3) using the genomic DNA of the strain to be tested as a template, performing PCR amplification and sequencing using degenerate primers; (4) using the Blastx tool in NCBI to compare the sequencing results with known sequences; and (5) testing the MC degradation ability of strains with positive gene alignment results to verify the effectiveness of the PCR screening method.

[0022] The test results showed that all the positive strains obtained by PCR amplification could effectively degrade MC, indicating that this method has a high accuracy rate.

[0023] The present invention also provides the use of the degenerate primer pair or the kit or the reagent in identifying or assisting in identifying microcystin-degrading bacteria.

[0024] The present invention also provides the use of the degenerate primer pair in preparing a product for identifying or assisting in identifying microcystin-degrading bacteria.

[0025] The mlr pathway is the most well-studied MC biodegradation pathway and is widely present in MC-degrading bacteria. Among them, the metalloprotease encoded by the mlrA gene is the initial degradation enzyme of this enzymatic reaction pathway. This gene is widely used as a molecular marker gene to judge whether a strain has MC degradation performance. Based on this molecular biology, universal primers can be designed for the mlrA gene, and PCR amplification and sequencing techniques can be used to perform preliminary identification of MC-degrading bacteria, and then the degradation ability of positive strains can be confirmed by liquid chromatography-mass spectrometry.

[0026] The purpose of the present invention is to provide a pair of universal degenerate primers for amplifying the MC degradation gene mlrA, thereby identifying MC degradation bacteria at the molecular level; the method provided by the present invention does not rely on batch shake flask experiments, and can achieve the purpose of simplifying screening steps, saving time and saving costs.

[0027] In order to quickly and accurately detect whether a sample to be tested contains microcystin-degrading bacteria, the present invention obtains a total of 41 published mlrA gene sequences from the NCBI database, obtains two gene conserved regions through sequence comparison analysis, and designs a pair of universal degenerate primers mlrA-354F and mlrA-785R for the mlrA gene, and establishes a method for quickly screening and identifying microcystin-degrading bacteria using the degenerate primer pair. When designing primers, the inventor covers all published mlrA gene sequences as much as possible, thereby ensuring the universality of the degenerate primers, and when designing primers, two upstream conserved regions and downstream conserved regions with a long distance are selected, thereby making the amplification product longer and the sequence comparison result more reliable. The designed primers can accurately identify positive bacteria with efficient MC degradation ability.

[0028] In one embodiment of the present invention, a reagent (Lysis Buffer for Microorganism to Direct PCR, TaKaRa Code No. 9164) is also used that can quickly lyse most bacteria to release nucleic acids, which can then be directly used in various PCR detection reactions. This method only requires taking a single clone of the strain to be tested and placing it in a lysis buffer at 80°C for 15 minutes, and then taking the DNA supernatant of the lysis buffer for subsequent PCR amplification, eliminating the complex operation of extracting genomic DNA, greatly saving time and cost.

[0029] Experiments have shown that the present invention has the following advantages compared with the prior art:

[0030] 1) Short screening cycle: The PCR screening method used in the present invention can obtain identification results within two days, while the traditional screening method requires at least one week. This method effectively shortens the screening cycle.

[0031] 2) Low screening cost: The screening of degrading bacteria in the present invention does not rely on batch shake flask experiments, thereby saving a large amount of expensive MC pure products and reducing the screening workload, greatly saving manpower, material resources and financial costs.

[0032] 3) Good screening effect: The specific implementation results show that the positive strains identified by the method provided by the present invention all have excellent MC degradation ability, indicating that the method has a good screening effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 is the degenerate primer sequence of mlrA gene.

[0034] Figure 2 The results of PCR product electrophoresis. M is DNA marker DL2000; ERW19, ERS04, ERS09, ERN02, ERN07, ERR01, and ERR08 represent the PCR amplification products of the seven strains to be tested, among which the amplified bands of ERW19 and ERN07 are of correct size.

[0035] Figure 3 These are the MC degradation curves of strains ERW19 and ERN07. LR represents microcystin MC-LR, and RR represents microcystin MC-RR. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0037] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0038] In the following examples, the test strains, reagents and instruments involved are:

[0039] 1. The 7 strains of bacteria used in the experiment were ERW19, ERS04, ERS09, ERN02, ERN07, ERR 01, and ERR08 (Yan-Hua Zeng, Zhong-Hua Cai, Jian-Ming Zhu, Xiao-Peng Du, Jin Zhou. Two hierarchical LuxR-LuxI type quorum sensing systems in Novosphingob iumactivate microcystin degradation through transcriptional regulation of themlr pathway. Water Research, 2020, 183: 116092), isolated from Taihu Lake water samples during the outbreak of cyanobacterial blooms. All test strains can be obtained from the Shenzhen International Graduate School of Tsinghua University, i.e., the applicant, to repeat the experiments of the present invention.

[0040] 2. The reagents and instruments used in the experiment include:

[0041] ① DNA lysis reagent: Lysis Buffer for Microorganism to Direct PCR (TaKaRa Code No. 9164);

[0042] ②PCR amplification reagents: Premix Taq TM (Ex Taq TM Version 2.0plus dye)(TaKaRa CodeNo.RR902Q);

[0043] ③ Reagents for agarose gel electrophoresis: Ethidium bromide (EB) and 50×TAE buffer were purchased from Shanghai Shenggong Bioengineering Technology Service Co., Ltd., and the agarose used was Biowest Agarose (Spanish agarose);

[0044] ④ Reagents used in MC degradation verification experiments: MgSO4·7H2O, KH2PO4, K2HPO4, NaCl, CaCl2, ZnCl2, FeSO4·7H2O, MnCl2·4H2O, CuCl2·2H2O, etc. were purchased from Sinopharm Chemical Reagent Co., Ltd., and microcystin (MC-LR and MC-RR) was purchased from Taiwan Algae Research Co., Ltd.;

[0045] ⑤ LC-MS / MS detection reagents: acetonitrile and formic acid (both chromatographically pure) were purchased from Merck, Germany, and the chromatographic column was a C18 column (Atlantis T3, 2.1 mm×100 mm, 3.0 μm) purchased from Waters, USA.

[0046] Example 1 Primer design

[0047] A total of 41 published mlrA gene sequences were obtained from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) by combining literature research and database mining. The ClustalW tool built into the BioEdit software (version 7.0.5.2) was used to perform multiple sequence alignment on the above sequences, and two conserved gene regions were obtained. Based on this, a pair of universal degenerate primers mlrA-354F / mlrA-785R (such as Figure 1 It is expected that the pair of degenerate primers can be used to amplify a target gene fragment of about 450 bp, which can be used to identify or assist in the identification of microcystin-degrading bacteria. The primers were commissioned to be synthesized by Guangzhou Aiji Biotechnology Co., Ltd.

[0048] The degenerate primer pair of the present invention consists of primer mlrA-354F and primer mlrA-785R; the primer mlrA-354F is a single-stranded DNA molecule shown in SEQ ID No.1; the primer mlrA-785R is a single-stranded DNA molecule shown in SEQ ID No.2; the nucleotide sequence shown in SEQ ID No.1 and / or SEQ ID No.2 is as follows:

[0049] Upstream primer mlrA-354F: 5'-TTCYTTGYVTTCTTCGCGCTC-3'

[0050] Downstream primer mlrA-785R: 5'-CCCAGYTCGTTATGGATSGCGTGA-3'

[0051] In the above degenerate primers, Y=C or T, V=G, A or C, and S=C or G.

[0052] The primers designed by the present invention cover all the published mlrA gene sequences as much as possible, thereby ensuring the universality of the degenerate primers, and two upstream conserved regions and downstream conserved regions with long distances are selected when designing the primers, thereby making the amplification product longer and the sequence comparison result more reliable.

[0053] Example 2 Method for screening microcystin-degrading bacteria (MC) using degenerate primers

[0054] 1. Obtaining DNA of the strain to be tested

[0055] The seven strains of bacteria used in the experiment were isolated from water samples of Taihu Lake during the outbreak of cyanobacteria blooms, and were numbered: ERW19, ERS04, ERS09, ERN02, ERN07, ERR01, and ERR08. DNA was obtained using a reagent that can quickly lyse most bacteria to release nucleic acids, which can then be directly used in various PCR detection reactions. The specific steps are:

[0056] ① Add 50 μL DNA lysis buffer (Lysis Buffer for Microorganism to Direct PCR) to each of the seven sterilized 200 μL centrifuge tubes;

[0057] ② Use a sterilized pipette tip to pick up single colonies of 7 strains to be tested, place them in 7 200μL centrifuge tubes, blow them gently a few times and then take them out;

[0058] ③ Place the 7 200 μL centrifuge tubes in step ② in a PCR instrument for heat denaturation at 80°C for 15 minutes, centrifuge at 4000 rpm for 1 minute, and then take 2 μL of the supernatant after lysis (DNA lysis supernatant) as the DNA template for PCR reaction.

[0059] 2. PCR amplification of mlrA degradation gene

[0060] The DNA lysate supernatant of the seven strains of bacteria to be tested obtained in step 1 was used as a DNA template for PCR amplification, and the PCR amplification reaction was carried out using degenerate primers mlrA-354F and mlrA-785R.

[0061] The PCR reaction system was as follows: 2 μL of DNA lysis supernatant, 0.2 μM each of upstream primer mlrA-354F and downstream primer mlrA-785R, 25 μL of Premix Taq, and sterile distilled water to make up to 50 μL.

[0062] The PCR reaction conditions were as follows: pre-denaturation, 95°C, 5 min; denaturation, 98°C, 10 s; annealing, 50°C, 30 s; extension, 72°C, 1 min; 30 cycles of denaturation, annealing and extension; continued extension, 72°C, 10 min, and finally maintained at 12°C until taken out to obtain the PCR amplification product.

[0063] 3. Detection of PCR products by agarose gel electrophoresis

[0064] ① Prepare agarose gel: weigh 0.5g agarose and place it in 50mL 1×TAE buffer (concentration of 1%), heat it in a microwave until the agarose is completely melted, cool it to 50℃-60℃ at room temperature, add ethidium bromide (EB) to a final concentration of 0.5μg / mL, then slowly pour the gel solution into the gel-making tank (insert a 1cm comb in advance), and gently remove the comb after the gel is completely solidified.

[0065] ② Sample loading and electrophoresis: Add 5 μL DNA marker and PCR product to each sample well in turn, turn on the electrophoresis instrument to start electrophoresis, control the voltage to maintain at around 120 V, and stop electrophoresis when the bromophenol blue band moves to about 2 cm from the front of the gel.

[0066] ③Observe and take pictures: Open the gel imager software, set various parameters, place the gel after electrophoresis on the UV projection platform of the gel imager, perform imaging analysis, and save the imaging results in picture format.

[0067] The results are as follows Figure 2 As shown, the PCR amplification products of strains ERW19 and ERN07 were both about 450 bp in size, which was consistent with theoretical predictions. The PCR products of these two strains were sent to Guangzhou Aiki Biotechnology Co., Ltd. for sequencing.

[0068] 4. Use Blastx tool for sequence alignment

[0069] ① According to the sequence peak map provided by the sequencing company, the amplified product sequences of strains ERW19 and ERN07 were read, which were SEQ ID No.3 and SEQ ID No.4, respectively, with a size of 449 bp.

[0070] ② Use the Blastx tool in NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to compare the sequencing results with the known sequences.

[0071] According to the Blastx comparison results, the amplified product sequences of strains ERW19 and ERN07 were 100% similar to the mlrA gene of the reported MC-degrading strain Novosphingobium sp. THN1. Therefore, two positive MC-degrading bacteria were screened and identified by PCR amplification using this pair of degenerate primers.

[0072] 5. Verification of MC degradation ability of positive strains ERW19 and ERN07

[0073] Further shake flask culture experiments were performed to verify that both positive strains (ERW19 and ERN07) had efficient MC degradation capabilities, indicating that the molecular biological method provided by the present invention has good screening effect and high accuracy. The main implementation steps and test results are as follows:

[0074] ① Shake flask experiment: Two positive bacteria strains ERW19 and ERN07 were inoculated into MSM (MgSO4·7H2O 1.0g / L; KH2PO4 0.5g / L; K2HPO4 4.0g / L; NaCl1.0g / L; CaCl2 20.0mg / L; FeSO4 5.0mg / L; ZnCl2 5.0mg / L; MnCl2·4H2O 5.0 mg / L; CuCl20.5mg / L) medium with MC (MC-LR and MC-RR concentrations were both 100μg / L) as the sole carbon source, and placed in a constant temperature shaker for shaking culture. The bacterial solution was taken every 6h to determine the remaining MC content in the culture medium;

[0075] ② LC-MS / MS determination of MC content: LC-MS / MS is composed of a high performance liquid chromatograph (LC-20A, Shimadzu, Japan) and a triple quadrupole tandem mass spectrometer (API 3200, AB, USA). The high performance liquid chromatograph is equipped with an automatic sampling system, the chromatographic column is a C18 column (Atlantis T3, 2.1 mm×100 mm, 3.0 μm, Waters, USA), the column temperature is 40°C, the mobile phase A is an ultrapure aqueous solution containing 0.1% formic acid (a solution composed of a solute and a solvent, the solute is formic acid, the solvent is ultrapure water, and the volume percentage of formic acid is 0.1% ultrapure aqueous solution), and the mobile phase B is an acetonitrile solution containing 0.1% formic acid (a solution composed of a solute and a solvent, the solute is formic acid, the solvent is acetonitrile, and the volume percentage of formic acid is 0.1%), and a linear gradient elution mode is used. ESI-MS / MS used a positive ion ESI ionization source and multiple reaction monitoring (MRM) mode.

[0076] The results are as follows Figure 3 As shown, both positive bacteria have efficient MC degradation ability, among which strain ERW19 can completely degrade two microcystins MC-LR and MC-RR within 12 hours; strain ERN07 can also degrade two microcystins by about 90% within 18 hours. It shows that the degenerate primers designed and screened by the present invention and the method for rapid screening and identification of microcystin degrading bacteria established by using the degenerate primers can accurately, simply and quickly identify efficient microcystin degrading bacteria, and the obtained MC degrading bacteria have higher degradation ability and can meet the needs of practical applications.

[0077] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims. SEQUENCE LISTING <110> Tsinghua University Shenzhen International Graduate School <120> Degenerate primers for rapid screening and identification of microcystin-degrading bacteria and their application <160> 4 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> Artificial sequence <400> 1 ttcyttgyvt tcttcgcgct c 21 <210> 2 <211> twenty four <212> DNA <213> Artificial sequence <400> 2 cccagytcgt tatggatsgc gtga 24 <210> 3 <211> 449 <212> DNA <213> Artificial sequence <400> 3 ttccttgcat tcttcgcgct cacaggaatc atgtgggttc agacatacct ctacgctccg 60 cctggtacgc ttgatcgtac cttcctgcgc tatgggtcag atccggtcgc catttatgtg 120 atgctggcag catcgctgct actcagccct ggcccgctgc tcgaagaact gggctggcgc 180 ggctttgcgc tgccgcagct cctcaagaag tttgaccccc ttaccgcagc ggtgatcctc 240 ggcatcatgt ggtgggcctg gcatttgcca cgcgacctgc caacactgtt ctccggcgcc 300 cctggcgcgg cctggagcgt tattgtcaaa caactcgtca tcgctcctgg gttcattgcg 360 agcaccatca tcgctgtctt cgtatgcaac aagctcggtg gatcaatgtg ggggggcgtg 420 ctcactcacg ccatccataa cgagctggg 449 <210> 4 <211> 449 <212> DNA <213> Artificial sequence <400> 4 ttccttgcat tcttcgcgct cacaggaatc atgtgggttc agacatacct ctacgctccg 60 cctggtacgc ttgatcgtac cttcctgcgc tatgggtcag atccggtcgc catttatgtg 120 atgctggcag catcgctgct actcagccct ggcccgctgc tcgaagaact gggctggcgc 180 ggctttgcgc tgccgcagct cctcaagaag tttgaccccc ttaccgcagc ggtgatcctc 240 ggcatcatgt ggtgggcctg gcatttgcca cgcgacctgc caacactgtt ctccggcgcc 300 cctggcgcgg cctggagcgt tattgtcaaa caactcgtca tcgctcctgg gttcattgcg 360 agcaccatca tcgctgtctt cgtatgcaac aagctcggtg gatcaatgtg ggggggcgtg 420 ctcactcacg ccatccataa cgagctggg 449

Claims

1. A method for identifying or assisting in identifying microcystin-degrading bacteria, characterized in that: The method comprises the following steps: A1) Extract DNA from the sample to be tested; A2) using the DNA of the sample to be tested as a template and using a degenerate primer pair to perform PCR amplification; determining whether the sample to be tested contains microcystin-degrading bacteria or whether the sample to be tested is microcystin-degrading bacteria according to whether the PCR amplification product contains a DNA fragment of 449 bp in size; The degenerate primer pair consists of primer mlrA-354F and primer mlrA-785R; the primer mlrA-354F is a single-stranded DNA molecule shown in SEQ ID No.1; the primer mlrA-785R is a single-stranded DNA molecule shown in SEQ ID No.

2.

2. The method according to claim 1, characterized in that: The method for determining whether the sample to be tested contains microcystin-degrading bacteria or whether the sample to be tested is microcystin-degrading bacteria according to whether the PCR amplification product contains a DNA fragment with a size of 449 bp is as follows: if the PCR amplification product contains a DNA fragment with a size of 449 bp, the sample to be tested contains microcystin-degrading bacteria or the sample to be tested is microcystin-degrading bacteria; if the PCR amplification product does not contain a DNA fragment with a size of 449 bp, the sample to be tested does not contain microcystin-degrading bacteria or the sample to be tested is not microcystin-degrading bacteria.

3. The method according to claim 1 or 2, characterized in that: The primer annealing condition for the PCR amplification is annealing at 50° C. for 30 seconds.

4. The method according to claim 1 or 2, characterized in that: The PCR amplification reaction procedure is: pre-denaturation at 95°C for 5 min; denaturation at 98°C for 10 s, annealing at 50°C for 30 s, extension at 72°C for 1 min, 30 cycles; and further extension at 72°C for 10 min.

5. The method according to claim 1 or 2, characterized in that: The method for extracting DNA from a sample to be tested comprises: placing the sample to be tested in a DNA lysis solution and reacting at 80° C. for 15 minutes.

6. Application of degenerate primer pairs in identification or auxiliary identification of microcystin-degrading bacteria; The degenerate primer pair consists of primer mlrA-354F and primer mlrA-785R; the primer mlrA-354F is a single-stranded DNA molecule shown in SEQ ID No.1; the primer mlrA-785R is a single-stranded DNA molecule shown in SEQ ID No.

2.

7. Application of degenerate primer pairs in the preparation of products for identification or auxiliary identification of microcystin-degrading bacteria; The degenerate primer pair consists of primer mlrA-354F and primer mlrA-785R; the primer mlrA-354F is a single-stranded DNA molecule shown in SEQ ID No.1; the primer mlrA-785R is a single-stranded DNA molecule shown in SEQ ID No.2.

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