Primer for amplifying ammonia monooxygenase gene of nitrospirillum ammonia oxidizing bacteria and application of primer

Abstract

The invention provides a primer for amplifying an ammonia monooxygenase gene of nitrospirillum ammonia oxidizing bacteria and application of the primer, and relates to the field of environmental microorganisms and ecology. According to the present invention, the primer has good specificity and good coverage, and compared with the classic primer amoA-1F / amoA-2R for amplification of the ammonia oxidizing bacteria amoA gene, the primer S1F / S1R can specifically amplify the amoA gene band of the Nitrosospira ammonia oxidizing bacteria, and does not amplify the amoA gene band of the Nitrosomonas ammonia oxidizing bacteria, such that the primer S1F / S1R can specifically amplify the amoA gene band of the Nitrosomonas ammonia oxidizing bacteria. In addition, the primer has very high sensitivity, and a plasmid standard product with the copy number concentration as low as 12.2 copies / [mu] L can also amplify a strip. The primer shows a good amplification effect in application, and can provide accurate guidance for research of nitrospirillum ammonia oxidizing bacteria.

Description

Technical Field

[0001] The present invention relates to the field of environmental microorganisms and ecology, and particularly relates to a primer for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira and its application. Background Art

[0002] Common nitrification can be divided into two steps: the ammonia oxidation process and the nitrite oxidation process. Among them, the ammonia oxidation process, as the rate-limiting step of nitrification, is crucial for the global nitrogen cycle. The ammonia oxidation process is mainly carried out by ammonia-oxidizing bacteria (AOB) through ammonia monooxygenase to convert ammonia into hydroxylamine, and then hydroxylamine is converted into nitrite through hydroxylamine oxidoreductase. Currently, a total of three genera of ammonia-oxidizing bacteria have been isolated and classified into the class Proteobacteria, among which Nitrosomonas and Nitrosospira belong to the β-proteobacteria subclass, and Nitrosococcus belongs to the γ-proteobacteria subclass. Currently, the ammonia-oxidizing bacteria isolated and purified in laboratories around the world are mainly of the genus Nitrosomonas, and there are few isolations and purifications of the genus Nitrosospira. The understanding of this group of ammonia-oxidizing bacteria mainly still relies on the genus Nitrosomonas. However, there is already a lot of evidence indicating that the relative abundance of the genus Nitrosospira is higher in natural environments, and it plays a major role in driving nitrification reactions, the generation of greenhouse gas N 2 O and the entire global nitrogen cycle process. Therefore, new methods need to be developed to detect and quantify AOB of the genus Nitrosospira in order to evaluate their contribution to the ammonia oxidation process and their functional relationship with other nitrifying microorganisms.

[0003] Ammonia monooxygenase (AMO) is a key enzyme for ammonia-oxidizing bacteria to carry out the nitrification process. The gene encoding the AMO subunit A (amoA) is a functional and phylogenetic marker gene widely used in the research of ammonia-oxidizing bacteria. The amoA gene sequence of AOB has a certain degree of conservation, and there are usually only one or two copies in the genome. Therefore, the number of AOB can be indirectly reflected based on the number of amoA genes. However, the classic primers amoA-1F / amoA-2R for the AOB functional gene amoA are not genus-specific in design. Therefore, if we focus on the enrichment, isolation, quantification, and qualitative research of AOB of the genus Nitrosospira, a pair of specific primers need to be designed to provide more accurate guidance.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] In order to overcome the deficiencies of the prior art, the object of the present invention is to provide a primer for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira (Nitrosospira).

[0006] Another object of the present invention is to provide the application of the above primers in the detection, quantification, and amplicon high-throughput sequencing analysis of ammonia-oxidizing bacteria of the genus Nitrosospira.

[0007] The object of the present invention is achieved by the following technical solutions:

[0008] A primer for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira (Nitrosospira) has the following sequence:

[0009] S1F: 5'-TSGTSATGGACACCGTCHTGCT-3';

[0010] S1R: 5'-CCCTCKGSAAASCCTTCTTCGCC-3';

[0011] The length of the specific band amplified by the primer for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira is 422 bp.

[0012] The nucleotide sequence of the primer for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira contains degenerate bases, where S refers to the base at this position being an equal proportion of G and C, H refers to the base at this position being an equal proportion of A, T, and C, and K refers to the base at this position being an equal proportion of G and T.

[0013] Application of the above primer for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira in the preparation of a product for detecting ammonia-oxidizing bacteria of the genus Nitrosospira.

[0014] The product includes a kit, test strip, chip, or high-throughput sequencing platform.

[0015] A kit contains the above primer for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira.

[0016] The application of the above primer or kit is one of the following applications:

[0017] (A) Application in the detection of ammonia-oxidizing bacteria of the genus Nitrosospira.

[0018] (B) Application in the quantitative detection of ammonia-oxidizing bacteria of the genus Nitrosospira.

[0019] (C) Application in the amplicon high-throughput sequencing analysis of ammonia-oxidizing bacteria of the genus Nitrosospira.

[0020] A method for detecting ammonia-oxidizing bacteria of the genus Nitrosospira includes the following steps:

[0021] (1) Extract the genomic DNA of the sample to be tested;

[0022] (2) Use the primers for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira to perform PCR amplification on the genomic DNA of the sample to be tested, and perform agarose gel electrophoresis. If the corresponding band is amplified, it proves that the sample to be tested contains ammonia-oxidizing bacteria of the genus Nitrosospira.

[0023] A method for quantitatively detecting ammonia-oxidizing bacteria of the genus Nitrosospira, comprising the following steps:

[0024] 1) Extract the genomic DNA of the sample to be tested;

[0025] 2) Use the primers for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira to perform PCR amplification on the genomic DNA of the sample to be tested, and perform agarose gel electrophoresis. Cut the gel, purify and recover the amplification product, construct a plasmid and clone and express it. Extract the plasmid, measure the nucleic acid concentration and calculate the copy number concentration, and then perform gradient dilution;

[0026] 3) Use the primers for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira to perform fluorescence quantitative PCR amplification on the plasmid, and establish a standard curve between the copy number concentration of the plasmid standard and the cycle number Ct value when reaching the fluorescence threshold;

[0027] 4) Use the primers for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira to perform fluorescence quantitative PCR amplification on the genomic DNA of the sample to be tested, substitute the result into the standard curve in step 3), and obtain the number of ammonia-oxidizing bacteria of the genus Nitrosospira in the sample to be tested.

[0028] A method for analyzing the diversity of ammonia monooxygenase gene amplicons of ammonia-oxidizing bacteria of the genus Nitrosospira by high-throughput sequencing, comprising the following steps:

[0029] (a) Extract the genomic DNA of the sample to be tested;

[0030] (b) Use the primers for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira to perform PCR amplification on the genomic DNA of the sample to be tested, and perform agarose gel electrophoresis. Cut the gel, purify and recover to obtain the amplification product;

[0031] (c) Perform high-throughput sequencing on the amplification product, and perform diversity analysis according to the sequencing results.

[0032] The present invention has the following advantages and effects compared with the prior art:

[0033] The present invention provides primers for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira and their applications. These primers have good specificity and coverage. Compared with the classical primers amoA-1F / amoA-2R for amplifying the amoA gene of ammonia-oxidizing bacteria, the primers S1F / S1R of the present invention can specifically amplify the amoA gene band of ammonia-oxidizing bacteria of the genus Nitrosospira, but not the amoA gene band of ammonia-oxidizing bacteria of the genus Nitrosomonas. In addition, these primers have high sensitivity, and plasmid standards with a copy number concentration as low as 12.2 copies / μL can also amplify bands. These primers show good amplification effects in applications and can provide precise guidance for the research on ammonia-oxidizing bacteria of the genus Nitrosospira. Description of the Drawings

[0034] Figure 1 It is a schematic diagram of the electrophoresis results of the PCR amplification products of the primers S1F / S1R for the enriched samples of ammonia-oxidizing bacteria of the genus Nitrosospira; among them, lanes 1-4: DNA of the first-generation enrichments of melon and fruit field soil using four enrichment methods respectively, lanes 5-8: DNA of the second-generation enrichments of melon and fruit field soil using four enrichment methods respectively, lanes 9-12: DNA of the first-generation enrichments of farmland soil using four enrichment methods respectively, lanes 13-16: DNA of the second-generation enrichments of farmland soil using four enrichment methods respectively, lanes 17-19: DNA of the first-generation enrichments of nut field soil using three enrichment methods respectively, lanes 20-22: DNA of the second-generation enrichments of nut field soil using three enrichment methods respectively; the four enrichment methods are in turn using ammonium salt as the substrate, using urea as the substrate, using both ammonium salt and urea as the substrate, using both ammonium salt and urea and adding the urease inhibitor NBPT as the substrate; the three enrichment methods are in turn using ammonium salt as the substrate, using urea as the substrate, using both ammonium salt and urea as the substrate; lane M: DL2000 DNA Marker, lane N: negative control, lane P: Nitrosospira tenuis NV12 DNA of ammonia-oxidizing bacteria of the genus Nitrosospira, set as the positive control.

[0035] Figure 2It is a schematic diagram of the electrophoresis results of the PCR amplification products of the primer pair S1F / S1R and the classical primer pair amoA-1F / amoA-2R currently used for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria; among them, each lane is respectively lane A: ammonia-oxidizing bacteria Nitrosomonas nitrosa Nm90 of the genus Nitrosomonas, lane B: ammonia-oxidizing bacteria Nitrosomonas communis Nm2 of the genus Nitrosomonas, lane C: ammonia-oxidizing bacteria Nitrosomonas sp. SCUT-1 of the genus Nitrosomonas, lane D: ammonia-oxidizing bacteria Nitrosospira tenuis NV12 of the genus Nitrosospira, lane N: negative control, lane M: DL2000 DNA Marker.

[0036] Figure 3 It is a schematic diagram of the electrophoresis results of the PCR amplification products of the primer S1F / S1R for plasmid standard products with dilution gradient concentrations; among them, each lane is respectively lane A: plasmid standard product with a copy number concentration of 12200 copies / μL, lane B: plasmid standard product with a copy number concentration of 1220 copies / μL, lane C: plasmid standard product with a copy number concentration of 122 copies / μL, lane D: plasmid standard product with a copy number concentration of 12.2 copies / μL, lane E: plasmid standard product with a copy number concentration of 1.22 copies / μL, lane N: negative control, lane M: DL2000 DNA Marker.

[0037] Figure 4 It is a schematic diagram of the standard curve established between the copy number concentration of the plasmid standard product and the cycle number Ct value when reaching the fluorescence threshold during the fluorescence quantitative PCR amplification with the primer S1F / S1R.

[0038] Figure 5 It is a schematic diagram of the melting curve during the fluorescence quantitative PCR amplification of the plasmid standard product with the primer S1F / S1R.

[0039] Figure 6 It is a schematic diagram of the phylogenetic tree constructed with the representative sequences OUT (read counts accounting for more than 1% of the total read counts of their respective samples) of the amplicons of 2 soil samples analyzed by high-throughput sequencing with the primer S1F / S1R. Detailed implementation mode

[0040] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0041] For the test methods without specific experimental conditions noted in the following examples, they are generally in accordance with conventional experimental conditions or the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are reagents and materials obtained through commercial channels.

[0042] Nitrosomonas nitrosa Nm90 was disclosed in the literature "Zhou L J, Han P, Yu Y, et al. Cometabolic biotransformation and microbial-mediated abiotic transformation of sulfonamides by three ammonia oxidizers[J]. Water Research, 2019, 159(AUG.1): 444 - 453.";

[0043] Nitrosomonas communis Nm2 was disclosed in the literature "Kozlowski J A, Kits K D, Stein LY. Genome Sequence of Nitrosomonas communis Strain Nm2, a Mesophilic Ammonia-Oxidizing Bacterium Isolated from Mediterranean Soil[J]. Genome Announcements, 2016, 4(1).";

[0044] Nitrosomonas sp. SCUT-1 was disclosed in the literature "Wu J, Zhan M, Yuan L, et al. Sealing solid agar in serum bottles for rapid isolation and long-term preservation of chemoautotrophic ammonia-oxidizing bacteria[J]. Water Research, 2024, 260(000): 9.";

[0045] Nitrosospira tenuis NV12 was disclosed in the literature "Issifu S, Acharya P, Kaur-Bhambra J, et al. Biological Nitrification Inhibitors with Antagonistic and Synergistic Effects on Growth of Ammonia Oxidisers and Soil Nitrification [J]. Microbial Ecology, 2024, 87(1): 1-11."

[0046] Example 1

[0047] Ammonia-oxidizing bacteria belong to uncultured and difficult-to-culture microorganisms and it is very difficult to form single colonies on solid media. Therefore, specific gene fragments are often amplified for qualitative and quantitative analysis. However, there are few reports on primers for ammonia-oxidizing bacteria of the genus Nitrosospira, so there is a lack of accurate methods to study this group. To solve this problem, based on previous experimental experience in the laboratory, the present invention designed a specific primer that can amplify ammonia-oxidizing bacteria of the genus Nitrosospira, as follows:

[0048] Sequences of 73 Nitrosomonas, 8 Nitrosococcus, and 22 AOB genomes of the genus Nitrosospira were downloaded from the NCBI official website. The corresponding sequences of the amoA gene were found and aligned in the MEGA software. The sequences were translated into amino acid sequences, and conservative regions were selected to manually design degenerate primers for amplifying the amoA gene of the genus Nitrosospira, as shown in Table 1:

[0049] Table 1 Primer sequences and amplification information

[0050]

[0051] Note: The nucleotide sequences contain degenerate bases. Here, S means that the base at this position is an equal proportion of G and C, H means that the base at this position is an equal proportion of A, T, and C, and K means that the base at this position is an equal proportion of G and T.

[0052] Example 2 Verification experiment on the amplification effect of primers

[0053] According to the amplification principle of the degenerate primers designed in Example 1, the amplification of the amoA gene of ammonia-oxidizing bacteria of the genus Nitrosospira can be achieved. To verify its effect and accurately detect ammonia-oxidizing bacteria of the genus Nitrosospira in the enriched samples, an experiment was designed to amplify the genome of the enriched samples to verify the amplification effect of the primers. The specific steps are as follows:

[0054] (1) Use suction filtration to collect the cells of the Nitrosospira ammonia-oxidizing bacteria enrichment from natural soils (melon and fruit field soil (location: Xiancheng Town, Shantou City), farmland soil (location: Suishi Village, Guangzhou City), nut field soil (location: Plantation of South China Agricultural University, Guangzhou City)). Extract genomic DNA using the CTAB method for backup. Refer to the method in the literature "Zhan Manjun, Chu Yucan, Luo Jianfei, etc. Urea promotes ammonia oxidation in acidic soils by increasing the abundance of Nitrosospira [J]. Molecular Plant Breeding, 2024, 22(21): 7230-7240." to obtain a high-abundance enrichment of Nitrosospira ammonia-oxidizing bacteria, denoted as the enrichment sample.

[0055] (2) Use the primers S1F and S1R designed in Example 1 to perform PCR amplification on 24 samples (including enrichment samples 1-22, negative control N, and positive control P) to verify the specificity of the primers. The reaction system and reaction conditions are shown in Tables 2 and 3.

[0056] Table 2 PCR reaction system

[0057]

[0058] Table 3 PCR reaction conditions

[0059]

[0060] Perform 1% agarose gel electrophoresis on the PCR amplification products, with a loading volume of 4 μL per lane. The results are as Figure 1 shown. The experimental results show that S1F / S1R can effectively amplify the enrichment samples, and the bands are clear and bright.

[0061] Comparison and verification of the amplification effects between Example 3 and the primers amoA-1F / amoA-2R

[0062] The primers amoA-1F / amoA-2R (cited from the literature "The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. [J]. Applied & Environmental Microbiology, 1997, 63(12): 4704-12.") are currently widely used primers for amplifying the gene of the A subunit of ammonia monooxygenase in ammonia-oxidizing bacteria. These primers and the primers S1F / S1R of the present invention were selected to perform colony PCR amplification on three ammonia-oxidizing bacteria of the genus Nitrosomonas, namely Nitrosomonas nitrosa Nm90, Nitrosomonas communis Nm2, Nitrosomonas sp. SCUT-1, and an ammonia-oxidizing bacterium of the genus Nitrosospira, Nitrosospira tenuis NV12. The PCR reaction system and conditions of amoA-1F / amoA-2R were referred to the report of the primer source, and the amplification method of S1F / S1R was referred to Example 2.

[0063] The amplification products of the two sets of primers were subjected to 1% agarose gel electrophoresis, and the sample loading volume for each lane was 4 μL. The results are as Figure 2 shown. The experimental results showed that the primers amoA-1F / amoA-2R could amplify bands for all four ammonia-oxidizing bacteria, and could not distinguish between the genus Nitrosomonas and the genus Nitrosospira. However, the primers S1F / S1R only amplified clear and bright bands for the ammonia-oxidizing bacteria of the genus Nitrosospira, showing good specificity. It is suitable for the detection of ammonia-oxidizing bacteria of the genus Nitrosospira in samples.

[0064] Example 4 Verification experiment of primers for fluorescence quantitative PCR amplification

[0065] The genomic DNA of the enriched culture sample of the ammonia-oxidizing bacteria of the genus Nitrosospira described in Example 2 was subjected to PCR amplification using the primers S1F / S1R provided by the present invention. The amplification method was referred to Example 2, and then gel electrophoresis was performed. The band with a length of 422 bp was cut out, purified, and recovered.

[0066] Clone and express the recovered fragment using the TA Zero Background Fast Cloning Kit (Zhuangmeng) and DH5α competent cells (Zhuangmeng) according to the instructions of the manual. After culturing, collect the bacterial cells and extract the plasmid using the Fast Plasmid Miniprep Kit (Zhuangmeng). Measure the nucleic acid concentration of the plasmid with a spectrophotometer to be 75.83 ng / μL. Calculate the copy number concentration of the plasmid to be 1.22×10 10 copies / μL according to Equation 1, and then prepare the standard products by serial dilution at a 10-fold gradient to obtain plasmid standard products with a series of concentrations of 1.22×10 9 、1.22×10 8 、1.22×10 7 、1.22×10 6 、1.22×10 5 、1.22×10 4 、1.22×10 3 、1.22×10 2 、1.22×10 1 、1.22×10 0 copies / μL.

[0067] Equation 1: Calculation formula for the copy number concentration of plasmid standard products

[0068]

[0069] In the formula:

[0070] N is the copy number concentration of the plasmid (copies / μL);

[0071] C is the measured concentration of plasmid DNA (ng / μL);

[0072] L is the number of base pairs of 1 copy of plasmid DNA (bp / copy);

[0073] 660 is the Dalton of one base pair (Daltons / bp);

[0074] Daltons is the Dalton, a unit of mass. Approximately 1 g is 6.02×10 23 Daltons.

[0075] Refer to the amplification method in Example 2, and use the primers S1F / S1R to perform PCR amplification on the plasmid standard products (1.22×10 0 ~1.22×10 4 copies / μL). Perform 1% agarose gel electrophoresis on the amplification products, and load 4 μL of the sample in each lane. The results are as Figure 3As shown, the experimental results show that the primer pair S1F / S1R can detect a plasmid standard of 1.22×10 1 copies / μL, with high sensitivity. It is suitable for the highly sensitive detection of ammonia-oxidizing bacteria of the genus Nitrosospira in samples.

[0076] The plasmid standard (1.22×10 4 ~1.22×10 9 copies / μL) was amplified by Q-PCR on a fluorescence quantitative PCR instrument (Applied Biosystems TM QuantStudio1). Three parallel samples were set for each concentration of the standard. The amplification system is shown in Table 4, and the amplification conditions were as follows: pre-denaturation at 94°C for 4 min; amplification reaction at 94°C for 20 s, 58°C for 20 s, and 72°C for 40 s, for a total of 40 cycles; the melting curve reaction conditions were: 95°C for 15 s, 60°C for 60 s, and 95°C for 1 s.

[0077] Table 4 Q-PCR reaction system

[0078]

[0079] After amplification, a standard curve was established between the initial template number of the standard and the Ct value (the number of cycles when reaching the fluorescence threshold) by the QuantStudio TM Design&Analysis Software program. It can be seen that the standard curve has a good linear relationship, with R 2 being 0.998, the slope being -3.57, and the amplification efficiency being 90.589% (as Figure 4 shown), and the melting curve peak of the amplification is single (as Figure 5 shown), indicating good amplification specificity. Thus, the primer pair S1F / S1R and the reaction conditions meet the requirements of the Q-PCR test and can be used for the quantitative detection of ammonia-oxidizing bacteria of the genus Nitrosospira.

[0080] Example 5 Verification experiment of primers for amplicon high-throughput sequencing and amplicon quality control

[0081] To verify the accuracy of the amplification results of primers S1F / S1R for soil samples and whether the primers are suitable for the amplicon high-throughput sequencing analysis of the amoA gene of ammonia-oxidizing bacteria in the genus Nitrosospira, genomic DNA of two soil samples (melon and fruit field soil and farmland soil in Example 2) was amplified by PCR using primers S1F / S1R, and the method was referred to Example 2. After verifying the amplified products by gel electrophoresis, the target fragment with a length of 422 bp was recovered by cutting the gel and purification, and then sent to a commercial company to complete the construction of the sequencing library and amplicon high-throughput sequencing. After noise reduction and chimera removal of the sequencing data, non-redundant sequences were clustered into OTUs at 97% similarity, representative sequences with read counts accounting for more than 1% of the total read counts of their respective samples were selected, and a phylogenetic tree was constructed using MEGA 11. The results are as Figure 6 shown. The experimental results show that the amplicon representative sequences are clustered with the amoA gene reference sequences of the genus Nitrosospira, indicating that primers S1F / S1R can accurately amplify the amoA gene of ammonia-oxidizing bacteria in the genus Nitrosospira, verifying the high specificity of the primers, and also indicating that primers S1F / S1R are suitable for the experimental requirements of the amplicon high-throughput sequencing analysis of the amoA gene of ammonia-oxidizing bacteria in the genus Nitrosospira.

[0082] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A primer for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira, characterized in that: The sequences of the primers are as follows: S1F: 5'-TSGTSATGGACACCGTCHTGCT-3'; S1R: 5'-CCCTCKGSAAASCCTTCTTCGCC-3'.

2. Use of the primers according to claim 1 in preparing a product for detecting ammonia oxidizing bacteria of the genus Nitrosospira.

3. The use according to claim 2, characterized in that: The products include test kits, test strips, chips or high-throughput sequencing platforms.

4. A kit, characterized in that: Comprising the primer according to claim 1.

5. Use of the primer according to claim 1 or the kit according to claim 4 in the detection of ammonia oxidizing bacteria of the genus Nitrosospira.

6. Use of the primers according to claim 1 or the kit according to claim 4 in the quantitative detection of ammonia oxidizing bacteria of the genus Nitrosospira.

7. Use of the primers according to claim 1 or the kit according to claim 4 in high-throughput sequencing analysis of amplicons of ammonia-oxidizing bacteria of the genus Nitrosospira.

8. A method for detecting ammonia oxidizing bacteria of the genus Nitrosospira, characterized in that: The steps include: (1) Extracting genomic DNA from the sample to be tested; (2) Using the primers described in claim 1, PCR amplification is performed on the genomic DNA of the sample to be tested, and agarose gel electrophoresis is performed. If the corresponding band is amplified, it is proved that the sample to be tested contains ammonia oxidizing bacteria of the genus Nitrosospira.

9. A method for quantitative detection of ammonia oxidizing bacteria of the genus Nitrosospira, characterized in that: The steps include: 1) Extracting genomic DNA from the sample to be tested; 2) using the primers described in claim 1 to perform PCR amplification on the genomic DNA of the sample to be tested, and performing agarose gel electrophoresis, excising the gel to purify and recover the amplified product, constructing a plasmid and cloning and expressing it, extracting the plasmid to determine the nucleic acid concentration and calculate the copy number concentration, and then performing gradient dilution; 3) using the primers described in claim 1 to perform fluorescence quantitative PCR amplification on the plasmid, and establishing a standard curve between the copy number concentration of the plasmid standard and the cycle number Ct value when the fluorescence threshold is reached; 4) Using the primers described in claim 1 to perform fluorescent quantitative PCR amplification on the genomic DNA of the sample to be tested, substituting the result into the standard curve of step 3) to obtain the number of ammonia oxidizing bacteria of the genus Nitrosospira in the sample to be tested.

10. A method for analyzing the diversity of ammonia monooxygenase gene amplicons of ammonia-oxidizing bacteria of the genus Nitrosospira by high-throughput sequencing, characterized in that: The steps include: (a) extracting genomic DNA from the sample to be tested; (b) using the primers described in claim 1 to perform PCR amplification on the genomic DNA of the sample to be tested, and performing agarose gel electrophoresis, cutting the gel, purifying and recovering, to obtain an amplified product; (c) Perform high-throughput sequencing on the amplified products and conduct diversity analysis based on the sequencing results.

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