Primer for amplifying comammox ammonia monooxygenase gene and application thereof

Abstract

The application discloses a primer for amplifying a complete ammonia oxidation bacteria (Comammox) ammonia monooxygenase gene and application thereof. The primer can simultaneously amplify the ammonia monooxygenase A subunit gene of two branches of Clade A and Clade B. The primer provided by the application has good specificity, high accuracy and a proper length of the amplified fragment, and provides an accurate, convenient and reliable method for qualitative and quantitative detection of the complete ammonia oxidation bacteria and high-throughput sequencing analysis of the amplified subunit.

Description

Technical Field

[0001] The present invention relates to the fields of environmental microorganisms and ecology, and in particular to a primer for amplifying ammonia monooxygenase gene of complete ammonia oxidizing bacteria Comammox and application thereof. Background Art

[0002] Nitrification, the oxidation of ammonia to nitrate via nitrite, is a crucial process in the biogeochemical nitrogen cycle and plays a vital role in biological wastewater treatment and controlling nitrogen fertilizer loss in agricultural production. Nitrification has long been considered a collaborative process involving microorganisms that oxidize ammonia and nitrite separately. Complete ammonia oxidizers (Comammox) are a newly discovered class of microorganisms that can directly oxidize ammonia to nitrate. This discovery necessitates the development of new methods to detect and quantify Comammox to assess their contribution to nitrification and their functional relationship with other nitrifying microorganisms.

[0003] All currently discovered Comammox species are classified as belonging to the genus Nitrospira, a group of nitrite-oxidizing bacteria, and do not form a monophyletic branch. Therefore, traditional phylogenetic analysis based on 16S rRNA gene sequences cannot distinguish between complete ammonia oxidizers and nitrite-oxidizing bacteria. Ammonia monooxygenase (AMO) is a key enzyme in the nitrification process of complete ammonia oxidizers. The gene encoding AMO subunit A (amoA) is a widely used functional and phylogenetic marker gene in the study of ammonia-oxidizing bacteria and archaea. The amoA gene sequence of Comammox is relatively conserved, with a total length of approximately 900 base pairs and typically only one to two copies in the genome. Therefore, the amoA gene sequence can be used to analyze the diversity of Comammox, and the number of amoA genes can indirectly reflect the number of Comammox species. Specific primers designed for the amoA gene can be used for qualitative and quantitative detection of Comammox species and for diversity analysis using high-throughput sequencing. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a primer for amplifying the ammonia monooxygenase gene of the complete ammonia oxidizing bacterium Comammox.

[0005] Another object of the present invention is to provide the use of the above primers in the detection, quantification and amplicon high-throughput sequencing analysis of the complete ammonia oxidizing bacteria Comammox.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A primer for amplifying the ammonia monooxygenase gene of the complete ammonia oxidizing bacterium Comammox, comprising at least one of the following primer pairs:

[0008] F1: GACTGGGATTTCTGGNTNGAYTGG;

[0009] F2: TATMGGCAGCCATTYGGNGCRAC;

[0010] R1: ACCTCGATCATCCGGATRTAYTCNGG;

[0011] R2: GAMGAACTKCCCRAWYTGCCACCA;

[0012] The above primers are paired two by two to obtain primer pairs F1 / R1, F1 / R2, F2 / R1, and F2 / R2.

[0013] The primer for amplifying the Comammox ammonia monooxygenase gene is a degenerate primer.

[0014] The specific band length amplified by the primer for amplifying the Comammox ammonia monooxygenase gene is 359-576 bp; preferably, the specific band length amplified by the primer pair F1 / R1 is 467 bp, the specific band length amplified by the primer pair F1 / R2 is 576 bp, the specific band length amplified by the primer pair F2 / R1 is 359 bp, and the specific band length amplified by the primer pair F2 / R2 is 465 bp.

[0015] The nucleotide sequence of the primer for amplifying the Comammox ammonia monooxygenase gene contains a degenerate base, wherein N indicates that the base at this position is A, G, C, and T in equal proportions, Y indicates that the base at this position is C and T in equal proportions, R indicates that the base at this position is A and G in equal proportions, M indicates that the base at this position is A and C in equal proportions, K indicates that the base at this position is G and T in equal proportions, and W indicates that the base at this position is A and T in equal proportions.

[0016] The above-mentioned primer for amplifying the Comammox ammonia monooxygenase gene is used in the detection, quantification, and amplicon high-throughput sequencing analysis of Comammox.

[0017] The above-mentioned application can simultaneously cover Clade A and Clade B, two evolutionary branches of Comammox.

[0018] A method for detecting Comammox, comprising the following steps:

[0019] (1) extracting genomic DNA from the sample to be tested;

[0020] (2) using the primer pair for amplifying the Comammox ammonia monooxygenase gene to perform PCR amplification on the genomic DNA of the sample, and performing agarose gel electrophoresis, if a corresponding band is amplified, it is proved that the sample contains the Comammox.

[0021] A method for quantifying the Comammox, comprising the following steps:

[0022] (1) extracting the genomic DNA of the sample to be tested;

[0023] (2) using the primer pair for amplifying the Comammox ammonia monooxygenase gene to perform PCR amplification on the genomic DNA of the sample, and performing agarose gel electrophoresis, cutting and purifying the gel to recover the amplification product, constructing a plasmid and cloning and expressing, extracting the plasmid to determine the nucleic acid concentration and calculate the copy number concentration, and then performing gradient dilution;

[0024] (3) using the primer pair for amplifying the Comammox ammonia monooxygenase gene to perform fluorescent quantitative PCR amplification on the plasmid, establishing a standard curve between the copy number concentration of the plasmid standard and the cycle number Ct value when the fluorescence domain value is reached;

[0025] (4) using the primer pair for amplifying the Comammox ammonia monooxygenase gene to perform fluorescent quantitative PCR amplification on the genomic DNA of the sample, and substituting the results into the above-mentioned standard curve to obtain the number of Comammox in the sample.

[0026] A method for analyzing the diversity of the Comammox ammonia monooxygenase gene amplicon by high-throughput sequencing, comprising the following steps:

[0027] (1) extracting the genomic DNA of the sample to be tested;

[0028] (2) using the primer pair for amplifying the Comammox ammonia monooxygenase gene to perform PCR amplification on the genomic DNA of the sample, and performing agarose gel electrophoresis, cutting and purifying the gel to recover the amplification product;

[0029] (3) performing high-throughput sequencing on the amplification product, and performing diversity analysis according to the sequencing results.

[0030] The present application has the following advantages and effects relative to the prior art:

[0031] The application provides a primer for amplifying a complete ammonia-oxidizing bacteria Comammox ammonia monooxygenase gene and an application thereof, which can simultaneously amplify the amoA genes of Clade A and Clade B branches, greatly simplifying the operation steps in application; compared with other primers that can simultaneously amplify the two branches, the primer provided by the application shows better amplification effect, and more accurate detection results can be obtained in application. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 is a schematic diagram of a phylogenetic tree constructed by a complete ammonia-oxidizing bacteria amoA gene reference nucleotide sequence.

[0033] Figure 2 is a schematic diagram of the electrophoresis results of PCR amplification products of samples by four groups of primers designed by the application, wherein each lane is M: DL2000 DNA Marker, A: vegetable field soil, B: tea tree soil, C: fruit tree soil 1, D: fruit tree soil 2, E: tidal flat soil, F: flower soil, CK: negative control, and P: complete ammonia-oxidizing bacteria enrichment culture.

[0034] Figure 3 is a schematic diagram of the electrophoresis results of PCR amplification products of samples by three groups of primers comamoAF / comamoAR, A189Y / C576r / CA209f (amplified by nested PCR, A189Y / C576r is used for the first step of amplification, and CA209f / C576r is used for the second step of amplification), Ntsp-amoA162F / Ntsp-amoA359R and primer F1 / R1 that can simultaneously amplify the amoA genes of Clade A and Clade B branches, wherein each lane is M: DL2000 DNA Marker, A: vegetable field soil, B: tea tree soil, C: fruit tree soil 1, D: fruit tree soil 2, E: tidal flat soil, F: flower soil, CK: negative control, and P: complete ammonia-oxidizing bacteria enrichment culture.

[0035] Figure 4 is a schematic diagram of a standard curve established between the copy number concentration of plasmid standard and the cycle number Ct value when the fluorescence domain value is reached when primer F1 / R1 is used for fluorescence quantitative PCR amplification.

[0036] Figure 5 is a schematic diagram of the melting curve when primer F1 / R1 is used for fluorescence quantitative PCR amplification of plasmid standard.

[0037] Figure 6 is a schematic diagram of a phylogenetic tree co-constructed by representative sequences ASV (read number accounting for more than 1% of the total read number of each sample) obtained by amplicon high-throughput sequencing analysis of six soil samples by primer F1 / R1 and 45 complete ammonia-oxidizing bacteria amoA gene reference sequences. DETAILED DESCRIPTION

[0038] The application will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the embodiments of the application are not limited thereto.

[0039] In the following embodiments, if no specific test conditions are specified, the test conditions are generally in accordance with the conventional test conditions or in accordance with the test conditions recommended by the reagent company. If no special instructions are given, the materials, reagents, etc. used are reagents and materials obtained from commercial channels.

[0040] Example 1

[0041] The complete ammonia-oxidizing bacteria Comammox belongs to uncultured and difficult-to-culture microorganisms, and there are few available reference amoA gene sequences, and the amoA gene sequences form two branches of Clade A and Clade B in systematics. Therefore, although some related primers have been reported, there are still the following problems in application:

[0042] (1) When using primers that can only amplify the amoA gene of Clade A or B branch to detect samples, two primers are needed to amplify and analyze the results after combining, which is cumbersome to operate;

[0043] (2) The known primers that can simultaneously amplify the amoA gene of Clade A and B branch have poor specificity, and when applied to sample PCR amplification detection, false positive or false negative results occur, and when used for fluorescence quantitative PCR (Q-PCR) amplification, the number of Comammox in the sample will be overestimated or underestimated;

[0044] (3) The length of the primer amplification sequence is too short, and the sequence information that can be obtained is limited, which is not suitable for application in PCR amplification sequencing classification identification and high-throughput sequencing analysis of Comammox diversity.

[0045] In order to solve the above problems, the present application designs a high-specificity primer that can simultaneously amplify the complete ammonia-oxidizing bacteria Comammox Clade A and B branch according to the laboratory's previous experimental experience, as follows:

[0046] The genome of the complete ammonia-oxidizing bacteria was retrieved and downloaded from NCBI, and a total of 45 amoA gene nucleotide sequences were obtained by searching the genome, and the sequences were imported into MEGA11 to construct a phylogenetic tree, as shown in Figure 1 The sequences cover two branches of Clade A and Clade B. The sequences were translated into amino acid sequences, and the conservative regions were selected to manually design degenerate primers for amplifying the amoA genes of the two branches, as shown in Table 1:

[0047] Table 1 Primer sequence and amplification information

[0048]

[0049] Note: The nucleotide sequence of the primer contains degenerate bases, where N refers to the bases at that position being A, G, C, and T in equal proportions, Y refers to the bases at that position being C and T in equal proportions, R refers to the bases at that position being A and G in equal proportions, M refers to the bases at that position being A and C in equal proportions, K refers to the bases at that position being G and T in equal proportions, and W refers to the bases at that position being A and T in equal proportions.

[0050] Example 2: Verification experiment of primer amplification effect

[0051] According to the amplification principle of the degenerate primers designed in Example 1, they can be paired in pairs to achieve amplification of the amoA gene of complete ammonia oxidizers. In order to verify their effectiveness and accurately detect complete ammonia oxidizers in complex soil samples, an experiment was designed to amplify the soil genome to verify the amplification effect of the primers. The specific steps are as follows:

[0052] (1) Collect 6 soil samples, including A: vegetable soil, B: tea soil, C: fruit tree soil 1, D: fruit tree soil 2, E: tidal flat soil, and F: flower soil;

[0053] (2) Take 0.5g soil sample and use Genomic DNA was extracted using the Soil DNA Kit (Omega) according to the manufacturer's instructions in triplicate, and the three genomic DNAs extracted from each soil sample were mixed into one for later use.

[0054] (3) Collect the cells of the enriched culture of complete ammonia oxidizing bacteria and extract genomic DNA using the CTAB method for future use. The enriched culture was obtained from the sludge of the water treatment plant based on the laboratory's previous work experience. The complete ammonia oxidizing bacteria containing the Clade A branch in the sample were verified by chemical activity determination and clone sequencing, and the sample was designated as the P group;

[0055] (4) The primers designed in Example 1 were paired in pairs, and PCR amplification was performed on seven samples using F1 / R1, F1 / R2, F2 / R1, and F2 / R2, respectively, 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]

[0059] Table 3 PCR reaction conditions

[0060] Step 1 94°C, 5 min Step 2 94℃，30s Step 3 60℃，30s Step 4 72℃，40s Step 5 Steps 2-4 repeated for 20 cycles with a decrease of 0.5°C in Step 3 temperature each cycle Step 6 94℃，30s Step 7 50℃，30s Step 8 72℃，40s Step 9 Steps 6-8 repeated for 15 cycles Step 10 72°C, 5 min

[0061] (5) The PCR amplification products were subjected to 1% agarose gel electrophoresis, with 4 μL of sample loaded into each lane. Figure 2 As shown in the figure, the experimental results showed that primer F1 / R2 failed to amplify environmental samples, proving that effective detection could not be achieved; among the other three primer groups, although the F2 / R2 and F2 / R1 groups could also achieve a certain amplification effect, the bands were not clear and some samples could not be amplified; the F1 / R1 group had the best amplification effect, and the amplified bands of all samples were clear and bright.

[0062] Comparison and verification of the amplification effect of Example 3 with other similar primers

[0063] Three groups of primers that have been reported in the literature to simultaneously amplify amoA genes of CladeA and CladeB branches were selected: comamoA F / comamoA R (cited from Abundance and community composition of comammox bacteria in different ecosystems by a universal primer set[J]. Science of the total environment, 2019, 691: 146-155.), A189Y / C576r / CA209f (cited from Ubiquity and diversity of complete ammonia oxidizers(comammox)[J]. Applied and Environmental Microbiology, 2018, 84(24): e01390-18. A189Y / C576r was used for the first step amplification, and CA209f / C576r was used for the second step amplification. The primers were Ntsp-amoA162F / Ntsp-amoA359R (cited from Comammox) Nitrospira are abundant ammonia oxidizers in diverse groundwater-fedrapid sand filter communities[J].Environmental microbiology,2018,20(3):1002-1015.) and the primers F1 / R1 provided by the present invention were used to perform PCR amplification on genomic DNA from the six soil samples and one enrichment culture sample described in Example 2. The sequences and amplification lengths of the three reported primer sets are shown in Table 4. The PCR reaction systems and conditions were the same as those reported in the primer sources. The amplification method for F1 / R1 was similar to that in Example 2.

[0064] Table 4 Sequences and amplification lengths of three sets of primers

[0065]

[0066] The amplified products of the four primer sets were subjected to 1% agarose gel electrophoresis, with 4 μL of sample loaded into each lane. Figure 3 As shown in the figure, the experimental results showed that the bands amplified by primers comamoA F / comamoA R and Ntsp-amoA162F / Ntsp-amoA359R were darker, and non-specific amplification occurred; the amplified band by primers A189Y / C576r / CA209f was bright, but non-specific amplification also occurred, and the primers were amplified in two steps by nested PCR, which was cumbersome and could not be used for fluorescent quantitative PCR; the amplified bands of primers F1 / R1 for all samples were single and bright, indicating that the primers and reaction conditions had good amplification effects and were suitable for PCR amplification to detect complete ammonia oxidizing bacteria in samples.

[0067] Example 4: Validation Experiment of Primers for Fluorescence Quantitative PCR Amplification

[0068] The genomic DNA of the complete ammonia oxidizing bacteria enriched culture sample described in Example 2 was amplified by PCR using the primers F1 / R1 provided by the present invention. The amplification method was referred to Example 2, and then gel electrophoresis was performed to purify and recover the 467 bp band.

[0069] The recovered fragments were cloned and expressed using the TA Zero Background Fast Cloning Kit (Zhuangmeng) and DH5α competent cells (Zhuangmeng) according to the instructions. After culture, the bacteria were collected and the plasmid was extracted using the Fast Plasmid Miniprep Kit (Zhuangmeng). The plasmid nucleic acid concentration was measured by spectrophotometer and was 154.652 ng / μL. The copy number concentration of the plasmid was converted to 6.0489×10 10 copies / μl, and then the standard was prepared by 10-fold serial dilution to obtain 6.0489×10 8 , 6.0489×10 7 , 6.0489×10 6 , 6.0489×10 5 , 6.0489×10 4 , 6.0489×10 3 Plasmid standards at a concentration of 200 copies / μl.

[0070] Formula 1: Calculation formula for the copy number concentration of plasmid standards

[0071]

[0072] Where:

[0073] N is the plasmid copy number concentration (copies / μL);

[0074] C is the measured plasmid DNA concentration (ng / μL);

[0075] L is the number of bases per copy of plasmid DNA (bp / copy);

[0076] 660 is one base pair of Daltons (Daltons / bp);

[0077] Daltons is a unit of mass, 1g is approximately 6.02×10 23 Daltons.

[0078] The plasmid standard was PCR-PCR-equipped with primers F1 / R1 in a fluorescent quantitative PCR instrument (Applied Biosystems TM Q-PCR amplification was performed on a QuantStudio 1). Three parallel samples were set for each concentration of standard. The amplification system is shown in Table 5. The amplification conditions were as follows: pre-deformation at 94°C for 4 min; amplification reaction at 94°C for 15 s, 60°C for 15 s, and 72°C for 34 s, for a total of 40 cycles; and melting curve reaction conditions were as follows: 95°C for 15 s, 60°C for 60 s, and 95°C for 1 s.

[0079] Table 5 Q-PCR reaction system

[0080]

[0081] After amplification, QuantStudio TM The Design & Analysis Software program automatically establishes a standard curve between the initial template number of the standard sample and the cycle number Ct value when the fluorescence threshold is reached. It can be seen that the standard curve has a good linear relationship, R 2 is 0.999, the slope is -3.552, and the amplification efficiency is 91.223% (e.g. Figure 4 ), and the amplified melting curve has a single peak (as shown Figure 5 As shown, the specificity of amplification is good. Thus, primers F1 / R1 and reaction conditions meet the requirements of Q-PCR test and can be used for the quantitative detection of complete ammonia oxidizing bacteria.

[0082] Example 5: Validation Experiment of Primers for Amplicon High-Throughput Sequencing and Quality Control of Amplification Products

[0083] To verify the accuracy of the amplification results obtained with primers F1 / R1 for soil samples and their suitability for high-throughput sequencing analysis of amoA gene amplicons from complete ammonia oxidizers, genomic DNA from the six soil samples described in Example 2 was amplified using primers F1 / R1, following the same procedures as in Example 2. The amplified products were verified by gel electrophoresis and then purified and recovered. The 467-bp target fragment was then sent to a commercial company for sequencing library construction and paired-end high-throughput sequencing of the amplicon using a Miseq PE300.

[0084] The high-throughput sequencing data were processed using the DADA2 plugin in Qiime2 for noise reduction, chimera filtering, and sequence splicing. Representative ASVs and feature tables were obtained for the amplicon high-throughput sequencing. A phylogenetic tree was constructed using the phylogeny plugin in Qiime2 using 213 representative sequences whose read counts accounted for at least 1% of the total read count in each sample, along with the 45 amoA gene sequences from the genome of a fully ammonia-oxidizing bacterium described in Example 1.

[0085] The results are as follows Figure 6 As shown, the experimental results showed that the representative sequences of the amplicon were clustered with the reference sequences of the amoA genes of the two branches, CladeA and CladeB, indicating that primers F1 / R1 can accurately and simultaneously amplify the amoA genes of complete ammonia oxidizing bacteria of the two branches, verifying the high specificity of the primers, and also indicating that primers F1 / R1 are suitable for the experimental requirements of high-throughput sequencing analysis of amplicons of the amoA genes of complete ammonia oxidizing bacteria.

[0086] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A primer for amplifying the ammonia monooxygenase gene of the complete ammonia oxidizing bacterium Comammox, characterized by: The nucleotide sequence of the primer is: F1: GACTGGGATTTCTGGNTNGAYTGG; R1: ACTCCGATCATCCGGATRTAYTCNGG; The primers are degenerate primers; The nucleotide sequence of the primer contains degenerate bases, wherein N means that the bases at that position are A, G, C and T in equal proportions, Y means that the bases at that position are C and T in equal proportions, and R means that the bases at that position are A and G in equal proportions.

2. The primer according to claim 1, wherein: The specific band obtained by amplification with the primers has a length of 467 bp.

3. Use of the primers according to claims 1 to 2 in the detection and / or quantification of the complete ammonia oxidizing bacterium Comammox.

4. Use of the primers according to claims 1 to 2 in high-throughput sequencing analysis of amplicons of the complete ammonia oxidizing bacterium Comammox.

5. The use according to claim 3 or 4, characterized in that: The application can simultaneously cover the two evolutionary branches of CladeA and CladeB of the complete ammonia oxidizing bacteria Comammox.

6. A method for detecting the complete ammonia oxidizing bacteria Comammox, characterized in that The steps include: (1) Extract genomic DNA from the sample to be tested; (2) Using the primers for amplifying the ammonia monooxygenase gene of the complete ammonia oxidizing bacteria Comammox as described in claims 1 and 2, the genomic DNA of the sample is amplified by PCR, and then subjected to agarose gel electrophoresis. If the corresponding band is amplified, it is proved that the sample contains the complete ammonia oxidizing bacteria Comammox.

7. A method for quantifying the complete ammonia oxidizing bacteria Comammox, characterized in that The steps include: (1) Extract genomic DNA from the sample to be tested; (2) Using the primers for amplifying the ammonia monooxygenase gene of the complete ammonia oxidizing bacteria Comammox as described in claims 1 to 2, PCR amplification is performed on the genomic DNA of the sample, and agarose gel electrophoresis is performed. The amplified product is purified and recovered by gel excision, a plasmid is constructed and cloned for expression, the plasmid is extracted, the nucleic acid concentration is determined, the copy number concentration is calculated, and then a gradient dilution is performed; (3) Using primers that amplify the ammonia monooxygenase gene of the complete ammonia oxidizing bacteria Comammox, the plasmid was amplified by fluorescence quantitative PCR, and a standard curve was established between the copy number concentration of the plasmid standard and the cycle number Ct value when the fluorescence threshold was reached; (4) Use primers that amplify the ammonia monooxygenase gene of the complete ammonia oxidizing bacteria Comammox to perform fluorescent quantitative PCR amplification on the genomic DNA of the sample, and substitute the results into the above standard curve to obtain the number of complete ammonia oxidizing bacteria in the sample.

8. A method for analyzing the diversity of ammonia monooxygenase gene amplicons of the complete ammonia oxidizing bacterium Comammox by high-throughput sequencing, characterized in that The steps include: (1) Extract genomic DNA from the sample to be tested; (2) Using the primers for amplifying the ammonia monooxygenase gene of the complete ammonia oxidizing bacteria Comammox as described in claims 1 and 2, PCR amplification is performed on the genomic DNA of the sample, and agarose gel electrophoresis is performed, and the gel is cut and purified to obtain an amplified product; (3) Perform high-throughput sequencing on the amplified products and perform diversity analysis based on the sequencing results.

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