A primer for amplifying the ammonia monooxygenase gene of nitrosospira ammonia-oxidizing bacteria and application thereof

Abstract

This invention provides primers for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria in the genus *Nitrosospira* and their applications, relating to the fields of environmental microbiology and ecology. These primers exhibit good specificity and coverage. Compared to the classic primers amoA-1F / amoA-2R for amplifying the amoA gene of ammonia-oxidizing bacteria, the primers S1F / S1R of this invention specifically amplify the amoA gene band of ammonia-oxidizing bacteria in the genus *Nitrosospira*, but not the amoA gene band of ammonia-oxidizing bacteria in the genus *Nitrosomonas*. Furthermore, these primers have high sensitivity, amplifying bands even with plasmid standards at copy numbers as low as 12.2 copies / μL. In application, these primers have demonstrated excellent amplification performance, providing precise guidance for the study of ammonia-oxidizing bacteria in the genus *Nitrosospira*.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology and ecology, and in particular to a primer for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira and its application. Background Technology

[0002] Nitrification typically involves two steps: ammonia oxidation and nitrite oxidation. Ammonia oxidation, as the rate-limiting step in nitrification, is crucial for the global nitrogen cycle. The ammonia oxidation process is primarily carried out by ammonia-oxidizing bacteria (AOBs) using ammonia monooxygenase to convert ammonia to hydroxylamine, which is then converted to nitrite by hydroxylamine oxidoreductase. Currently, three genera of ammonia-oxidizing bacteria have been isolated and classified in the class Proteobacteria: *Nitrosomonas* and *Nitrosospira* (belonging to the subclass β-Proteobacteria), and *Nitrosococcus* (belonging to the subclass γ-Proteobacteria). Currently, the ammonia-oxidizing bacteria isolated and purified in laboratories worldwide are mainly from the genus *Nitrosomonas*, with very few isolated and purified from the genus *Nitrosospira*. Therefore, our understanding of this group of ammonia-oxidizing bacteria is primarily based on *Nitrosomonas*. However, substantial evidence suggests that *Nitrosporobacter* spp. are relatively more abundant in the natural environment and play a major role in driving nitrification, the formation of the greenhouse gas N2O, and the overall Earth's nitrogen cycle. Therefore, new methods are needed to detect and quantify *Nitrosporobacter* AOBs to assess their contribution to ammonia oxidation and their functional relationships with other nitrifying microorganisms.

[0003] Ammonia monooxygenase (AMO) is a key enzyme in the nitrification process of ammonia-oxidizing bacteria. The gene encoding the AMO subunit A (amoA) is a widely used functional and phylogenetic marker gene in ammonia-oxidizing bacteria research. The amoA gene sequence of AOBs (ammonia-oxidizing bacteria) is somewhat conserved, typically containing only one or two copies in the genome. Therefore, the number of amoA genes can indirectly reflect the number of AOBs. However, the classic primers amoA-1F / amoA-2R for the functional gene amoA of AOBs are not genus-specific in their design. Therefore, if focusing on the enrichment, isolation, quantification, qualitative analysis, and related research of AOBs in the genus *Nitrostrophomonas*, a pair of specific primers needs to be designed to provide more precise guidance.

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

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the purpose of this invention is to provide primers for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrosospira.

[0006] Another objective of this 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 *Nitrostrophus*.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A primer sequence for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria in the genus *Nitrosospira* is as follows:

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

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

[0011] The specific band length obtained by the primers used to amplify the ammonia monooxygenase gene of *Nitrostrophus* was 422 bp.

[0012] The primers for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus *Nitrostrophus* have nucleotide sequences containing degenerate bases, where S indicates that the bases at that position are equal proportions of G and C, H indicates that the bases at that position are equal proportions of A, T, and C, and K indicates that the bases at that position are equal proportions of G and T.

[0013] The application of the primers described above for amplifying the ammonia monooxygenase gene of *Nitrostrophus* in the preparation of products for detecting *Nitrostrophus* ammonia-oxidizing bacteria.

[0014] The products include reagent kits, test strips, chips, or high-throughput sequencing platforms.

[0015] A kit containing primers for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus *Nitrosporium*.

[0016] The above primers or kits are used in one of the following applications:

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

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

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

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

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

[0022] (2) Using the primers described above for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus Nitrospilurus, the genomic DNA of the sample to be tested was amplified by PCR and subjected to agarose gel electrophoresis. If the corresponding band was amplified, it proved that the sample to be tested contained ammonia-oxidizing bacteria of the genus Nitrospilurus.

[0023] A quantitative detection method for ammonia-oxidizing bacteria of the genus *Nitrosporioides* includes the following steps:

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

[0025] 2) Using the primers described above for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus *Nitrostrophus*, PCR amplification was performed on the genomic DNA of the test sample. Agarose gel electrophoresis was then performed, the amplification product was excised and purified, a plasmid was constructed and cloned for expression, the plasmid was extracted, the nucleic acid concentration was measured and the copy number concentration was calculated, and then serial dilutions were performed.

[0026] 3) Use the primers described above for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus *Nitrostrophus* to amplify the plasmid by real-time quantitative PCR and establish a standard curve between the copy number concentration of the plasmid standard and the cycle number Ct value when the fluorescence threshold is reached.

[0027] 4) Using the primers described above for amplifying the ammonia monooxygenase gene of Nitrostrophus, perform real-time PCR amplification on the genomic DNA of the sample to be tested. Substitute the results into the standard curve in step 3) to obtain the number of ammonia-oxidizing bacteria of Nitrostrophus in the sample to be tested.

[0028] A method for analyzing the diversity of ammonia monooxygenase gene amplicones in ammonia-oxidizing bacteria of the genus *Nitrosporobacter* using high-throughput sequencing includes the following steps:

[0029] (a) Extracting genomic DNA from the sample to be tested;

[0030] (b) Using the primers described above for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria of the genus *Nitrostrophus*, the genomic DNA of the test sample was amplified by PCR, followed by agarose gel electrophoresis, gel excision and purification to obtain the amplified product.

[0031] (c) Perform high-throughput sequencing on the amplified products and conduct diversity analysis based on the sequencing results.

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

[0033] This invention provides primers for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria in the genus *Nitrosospira* and their applications. These primers exhibit good specificity and coverage. Compared to the classic primers *amoA-1F* / *amoA-2R* for amplifying the *amoA* gene in ammonia-oxidizing bacteria, the primers S1F / S1R of this invention specifically amplify the *amoA* gene band of *Nitrosospira* ammonia-oxidizing bacteria, but not the *amoA* gene band of *Nitrosomonas* ammonia-oxidizing bacteria. Furthermore, these primers demonstrate high sensitivity, amplifying bands even with plasmid standards at copy numbers as low as 12.2 copies / μL. In application, these primers have shown excellent amplification performance, providing precise guidance for the study of ammonia-oxidizing bacteria in the genus *Nitrosospira*. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the electrophoresis results of PCR amplification products of ammonia-oxidizing bacteria (Nitrostrophus spp.) enriched using primers S1F / S1R; lanes 1-4: first-generation enriched DNA from fruit and vegetable field soil using four enrichment methods; lanes 5-8: second-generation enriched DNA from fruit and vegetable field soil using four enrichment methods; lanes 9-12: first-generation enriched DNA from farmland soil using four enrichment methods; lanes 13-16: second-generation enriched DNA from farmland soil using four enrichment methods. Lanes 17-19: First-generation enriched DNA from nut field soils using three enrichment methods; Lanes 20-22: Second-generation enriched DNA from nut field soils using three enrichment methods; The four enrichment methods were, in order: ammonium salt as substrate, urea as substrate, simultaneous addition of ammonium salt and urea as substrate, and simultaneous addition of ammonium salt and urea with urease inhibitor NBPT as substrate; The three enrichment methods were, in order: ammonium salt as substrate, urea as substrate, and simultaneous addition of ammonium salt and urea as substrate; Lane M: DL2000 DNA Marker; Lane N: Negative control; Lane P: DNA of Nitrosospiratenuis NV12, used as a positive control.

[0035] Figure 2This is a schematic diagram of the electrophoresis results of PCR amplification products of four ammonia-oxidizing bacteria using primers S1F / S1R and the classic primers amoA-1F / amoA-2R currently used for amplifying the ammonia monooxygenase gene of ammonia-oxidizing bacteria. Lanes A, B, C, D, and M represent: Lane A: Nitrosomonas nitrosa Nm90; Lane B: Nitrosomonas communis Nm2; Lane C: Nitrosomonas sp. SCUT-1; Lane D: Nitrosospira tenuis NV12; Lane N: negative control; Lane M: DL2000 DNA Marker.

[0036] Figure 3 This is a schematic diagram of the electrophoresis results of PCR amplification products of PCR using primers S1F / S1R with varying concentrations of plasmid standards. Lanes A, B, C, D, E, and N represent the plasmid standards with a copy number of 12200 copies / μL, respectively. Lane N represents the negative control, and lane M represents the DL2000 DNA Marker.

[0037] Figure 4 This is a schematic diagram of the standard curve established between the copy number concentration of plasmid standard and the cycle number Ct value at which the fluorescence threshold is reached when primers S1F / S1R are used for quantitative real-time PCR amplification.

[0038] Figure 5 This is a schematic diagram of the melting curve of primers S1F / S1R when used for quantitative real-time PCR amplification of plasmid standards.

[0039] Figure 6 This is a schematic diagram of the phylogenetic tree constructed using the representative OUT sequences (more than 1% of the total reads in each sample) of primers S1F / S1R for high-throughput sequencing analysis of amplicon sequences from two soil samples. Detailed Implementation

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

[0041] Unless otherwise specified, the test methods in the following examples are generally performed under standard experimental conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used are commercially available.

[0042] Nitrosomonas nitrosa Nm90 is disclosed in the literature “Zhou LJ, 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 is disclosed in the document "Kozlowski JA, Kits KD, 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 is disclosed in the literature “Wu J,Zhan M,Yuan L,et al.Sealing solidagar 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 is disclosed in the document "Issifu S, Acharya P, Kaur-Bhambra J, etal. Biological Nitrification Inhibitors with Antagonistic and SynergisticEffects on Growth of Ammonia Oxidisers and Soil Nitrification[J]. Microbial Ecology, 2024, 87(1):1-11."

[0046] Example 1

[0047] Ammonia-oxidizing bacteria are difficult to culture and uncultured microorganisms, rarely forming single colonies on solid culture media. Therefore, amplification of their specific gene fragments is often used for qualitative and quantitative analysis. However, primers for ammonia-oxidizing bacteria of the genus *Nitrospirillum* are almost non-existent, resulting in a lack of precise methods for studying this population. To address this issue, based on previous laboratory experimental experience, this invention designs a specific primer capable of amplifying ammonia-oxidizing bacteria of the genus *Nitrospirillum*, as follows:

[0048] Sequences of 73 Nitrosomonas, 8 Nitrosococcus, and 22 Nitrosospira AOB genomes were downloaded from the NCBI website. The sequence corresponding to the amoA gene was identified and aligned using MEGA software. The sequences were translated into amino acid sequences, and conserved regions were selected. Degenerate primers for amplifying the Nitrosospira amoA gene were manually designed, as shown in Table 1.

[0049] Table 1 Primer sequences and amplification information

[0050]

[0051] Note: Nucleotide sequences contain degenerate bases, where S indicates that the bases at that position are equal proportions of G and C, H indicates that the bases at that position are equal proportions of A, T, and C, and K indicates that the bases at that position are equal proportions of G and T.

[0052] Example 2: Verification Experiment of Primer Amplification Effect

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

[0054] (1) The bacterial cells of the Nitrosospira genus ammonia-oxidizing bacteria were collected from natural soils (soils from fruit and vegetable fields (location: Xiancheng Town, Shantou City), farmland soils (location: Suishi Village, Guangzhou City), and nut field soils (location: South China Agricultural University Plantation, Guangzhou City)) by vacuum filtration. Genomic DNA was extracted using the CTAB method for later use. The high abundance of Nitrosospira genus ammonia-oxidizing bacteria was obtained by referring to the method in the literature "Zhan Manjun, Chu Yucan, Luo Jianfei, et al. Urea promotes ammonia oxidation in acidic soil by increasing Nitrosospira abundance [J]. Molecular Plant Breeding, 2024, 22(21):7230-7240.", and was recorded as the enriched sample.

[0055] (2) The primers S1F and S1R designed in Example 1 were used to amplify PCR on 24 samples (including enriched samples 1 to 22, negative control N, and positive control P) to verify the specificity of the primers. The reaction system and reaction conditions are shown in Table 2 and Table 3.

[0056] Table 2 PCR reaction system

[0057]

[0058] Table 3 PCR reaction conditions

[0059]

[0060] The PCR amplification products were subjected to 1% agarose gel electrophoresis, with 4 μL loaded onto each lane. The results are as follows: Figure 1 As shown in the figure, the experimental results show that S1F / S1R can effectively amplify enriched samples, and the bands are clear and bright.

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

[0062] 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 ammonia monooxygenase A subunit gene of ammonia-oxidizing bacteria. These primers, along with primers S1F / S1R of this invention, were used to perform colony PCR amplification on three ammonia-oxidizing bacteria of the genus *Nitrosomonas* nitrosa Nm90, *Nitrosomonas communis* Nm2, and *Nitrosomonas sp. SCUT-1*, and one ammonia-oxidizing bacterium of the genus *Nitrosospira tenuis* NV12. The PCR reaction system and conditions for amoA-1F / amoA-2R were as reported in the primer source, and the amplification method for S1F / S1R was as described in Example 2.

[0063] The amplification products of the two primer sets were subjected to 1% agarose gel electrophoresis, with a sample volume of 4 μL per lane. The results are as follows: Figure 2 As shown in the results, primers amoA-1F / amoA-2R amplified bands for all four ammonia-oxidizing bacteria strains, but could not distinguish between *Nitrosomonas* and *Nitrospirillum*. Primers S1F / S1R, however, amplified clear and bright bands only for *Nitrospirillum* ammonia-oxidizing bacteria, demonstrating good specificity. This method is suitable for detecting *Nitrospirillum* ammonia-oxidizing bacteria in samples.

[0064] Example 4: Validation experiment of primers for real-time PCR amplification

[0065] The genomic DNA of the enriched culture sample of ammonia-oxidizing bacteria of the genus *Nitrostrophus* described in Example 2 was amplified by PCR using the primers S1F / S1R provided by this invention. The amplification method was the same as in Example 2. Then, gel electrophoresis was performed, and the 422bp band was cut and purified by gel electrophoresis.

[0066] Following the instructions, the recovered fragment was cloned and expressed using the TA Zero Background Fast Cloning Kit (Zhuangmeng) and DH5α competent cells (Zhuangmeng). After culturing, bacterial cells were collected, and plasmids were extracted using the Fast Plasmid Miniprep Kit (Zhuangmeng). The plasmid nucleic acid concentration was measured to be 75.83 ng / μL using a spectrophotometer, and the copy number concentration of the plasmid was calculated to be 1.22 × 10⁻¹⁰ using Equation 1. 10 The concentration was increased to 1.22 × 10⁻¹¹ μL, and then a standard was prepared by serial dilution of 10-fold to obtain 1.22 × 10⁻¹¹ μL. 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 Plasmid standards with concentrations in copies / μL series.

[0067] Formula 1: Formula for calculating the copy number concentration of plasmid standards

[0068]

[0069] In the formula:

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

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

[0072] L represents the number of bases (bp / copy) in 1 copy of plasmid DNA;

[0073] 660 is one base pair in Daltons (Daltons / bp);

[0074] Daltons is a unit of mass; 1 g is approximately 6.02 × 10⁻⁶. 23 Daltons.

[0075] Referring to the amplification method in Example 2, primers S1F / S1R were used to amplify the plasmid standard (1.22 × 10⁻⁶). 0 ~1.22×10 4 PCR amplification was performed using copies / μL, and the amplification products were subjected to 1% agarose gel electrophoresis, with 4 μL loaded onto each lane. The results are as follows: Figure 3As shown, the experimental results indicate that primers S1F / S1R can detect 1.22 × 10⁻⁶. 1 The plasmid standard, with copies / μL, exhibits high sensitivity. It is suitable for highly sensitive detection of ammonia-oxidizing bacteria of the genus *Nitrosporium* in samples.

[0076] Primers S1F / S1R were used to test the plasmid standard (1.22 × 10⁻⁶). 4 ~1.22×10 9 (copies / μL) on a real-time PCR instrument (Applied Biosystems) TM Q-PCR amplification was performed on QuantStudio1. Three parallel samples were set for each concentration of standard. The amplification system is shown in Table 4. The amplification conditions were: 94℃ pre-deformation for 4 min; amplification reaction at 94℃ for 20 s, 58℃ for 20 s, and 72℃ for 40 s for a total of 40 cycles; the melting curve reaction conditions were: 95℃ for 15 s, 60℃ for 60 s, and 95℃ for 1 s.

[0077] Table 4. Q-PCR reaction system

[0078]

[0079] After amplification, by QuantStudio TM The Design & Analysis Software program generates a standard curve relating the initial template number to the cycle number Ct at which the fluorescence threshold is reached. The standard curve shows a good linear relationship, with R0... 2 The value was 0.998, the slope was -3.57, and the amplification efficiency was 90.589% (e.g., Figure 4 As shown), and the amplified melting curve has a single peak (as shown). Figure 5 As shown in the figure, the amplification specificity is good. Therefore, the primers S1F / S1R and the reaction conditions meet the requirements of Q-PCR and can be used for the quantitative detection of ammonia-oxidizing bacteria of the genus *Nitrosporium*.

[0080] Example 5: Validation experiment using primers for amplicon high-throughput sequencing and mass control of amplified products.

[0081] To verify the accuracy of primers S1F / S1R in amplifying soil samples and to determine their suitability for high-throughput sequencing analysis of amoA genes from *Nitrostrophobicella* spp. ammonia-oxidizing bacteria, genomic DNA from two soil samples (fruit and vegetable field soil and farmland soil from Example 2) was amplified using primers S1F / S1R, following the method described in Example 2. After gel electrophoresis verification, the amplified products were gel-cleaved and purified to recover the 422 bp target fragment, which was then sent to a commercial company for sequencing library construction and amplicon high-throughput sequencing. After noise reduction and chimera removal, the sequencing data were clustered into OTUs based on 97% similarity. Representative sequences representing more than 1% of the total reads in each sample were selected, and a phylogenetic tree was constructed using MEGA 11. The results are as follows: Figure 6 As shown in the figure, the experimental results indicate that the representative sequence of the amplicon clusters with the reference sequence of the amoA gene of the genus Nitrosospira, indicating that primers S1F / S1R can accurately amplify the amoA gene of ammonia-oxidizing bacteria of the genus Nitrosospira, verifying the high specificity of the primers, and also indicating that primers S1F / S1R are suitable for the experimental requirements of high-throughput sequencing analysis of amplicon of the amoA gene of ammonia-oxidizing bacteria of the genus Nitrosospira.

[0082] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

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

2. The use of the primers according to claim 1 in the preparation of products for detecting ammonia-oxidizing bacteria of the genus *Nitrosporium*.

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

4. A reagent kit, characterized in that, It contains the primers as described in claim 1.

5. The application of the primers of claim 1 or the kit of claim 4 in the detection of ammonia-oxidizing bacteria of the genus *Nitrosporium*.

6. The application of the primers of claim 1 or the kit of claim 4 in the quantitative detection of ammonia-oxidizing bacteria of the genus *Nitrostrophus*.

7. The application of the primers of claim 1 or the kit of claim 4 in high-throughput sequencing analysis of amplicon of ammonia-oxidizing bacteria of the genus *Nitrostrophus*.

8. A method for detecting ammonia-oxidizing bacteria of the genus *Nitrosporium*, characterized in that, Includes the following steps: (1) Extract genomic DNA from the sample to be tested; (2) Use the primers described in claim 1 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 Nitrospisil.

9. A method for quantitative detection of ammonia-oxidizing bacteria of the genus *Nitrosporobacter*, characterized in that, Includes the following steps: 1) Extract genomic DNA from the sample to be tested; 2) Using the primers described in claim 1, PCR amplification of the genomic DNA of the sample to be tested was performed, followed by agarose gel electrophoresis. The amplification product was then excised, purified, and recovered. A plasmid was constructed and cloned for expression. The plasmid was extracted, the nucleic acid concentration was measured, and the copy number concentration was calculated. Then, a serial dilution was performed. 3) Use the primers described in claim 1 to amplify the plasmid using real-time PCR and establish a standard curve between the plasmid standard copy number concentration and the cycle number Ct value when the fluorescence threshold is reached; 4) Use the primers described in claim 1 to perform real-time PCR amplification on the genomic DNA of the sample to be tested, and substitute the results into the standard curve of step 3) to obtain the number of ammonia-oxidizing bacteria of the genus Nitrosporium in the sample to be tested.

10. A method for analyzing the diversity of ammonia monooxygenase gene amplicones in ammonia-oxidizing bacteria of the genus *Nitrosporium* using high-throughput sequencing, characterized in that... Includes the following steps: (a) Extracting genomic DNA from the sample to be tested; (b) Using the primers described in claim 1, the genomic DNA of the sample to be tested was amplified by PCR, and then subjected to agarose gel electrophoresis, gel excision and purification to obtain the amplified product; (c) Perform high-throughput sequencing on the amplified products and conduct diversity analysis based on the sequencing results.

Images