Primers, kits, and PCR methods for detecting Methanomethylophilaceae in cellar mud of strong-aroma baijiu (Chinese liquor).

Abstract

This invention discloses primers, a kit, and a PCR method for detecting Methanomethylophilaceae in the cellar mud of strong-aroma baijiu (Chinese liquor), belonging to the field of microbial identification technology. This invention discloses a primer pair for the specific identification of Methanomethylophilaceae. The primer pair is specifically designed to target the methanol methyltransferase encoded by Methanomethylophilaceae. mtaB The primers, designed based on conserved gene sequences, include upstream and downstream primers. This invention utilizes these primer pairs and employs qPCR or PCR methods to specifically detect Methanomethylophilaceae in strong-aroma baijiu (Chinese liquor) cellar mud samples. This detection method is highly specific and sensitive, enabling accurate and rapid detection of Methanomethylophilaceae in cellar mud samples, providing technical support for optimizing the strong-aroma baijiu production process.

Description

Technical Field

[0001] This invention relates to the field of microbial identification technology, specifically to primers, kits, and PCR methods for detecting Methanomethylophilaceae in the cellar mud of strong-aroma baijiu. Background Technology

[0002] The unique flavor of strong-aroma baijiu is closely related to the complex and stable microbial community structure of its fermentation pit mud. As a unique anaerobic fermentation ecosystem, the pit mud is rich in bacteria and archaea. Methanomethylophilaceae, belonging to the Methanomaciphyllaceae order, is a newly discovered group of methanogenic archaea in recent years. Unlike most methanogenic archaea that utilize H2 / CO2 or acetic acid, this group can use methyl compounds such as methanol and methylamine as methanogenic substrates and may use hydrogen or extracellular electrons as electron donors. This unique metabolic characteristic suggests that Methanomethylophilaceae may have a dual potential function in the pit mud: firstly, by consuming substances such as methanol, it participates in the cycling of methyl compounds in the pit mud; secondly, by consuming hydrogen, it may relieve hydrogen feedback inhibition on acid-producing bacteria, thereby promoting the formation of key flavor precursors such as hexanoic acid.

[0003] However, physiological studies of Methanomethylophilaceae and exploration of its ecological functions in pit mud still face technical bottlenecks. The vast majority of these microorganisms are difficult to culture or uncultured, making it difficult to obtain pure cultures using traditional isolation and culture techniques, thus hindering the identification and counting of them using classical microbiological methods. Currently, the detection and identification of methanogenic archaea in environmental samples mainly relies on molecular biology techniques targeting the 16S rRNA gene. Although the 16S rRNA gene is widely used as a phylogenetic marker, its sequence conservation is high among closely related species, especially within certain groups of methanogenic archaea, resulting in limited resolution. Using universal or broad-spectrum primers may fail to accurately distinguish Methanomethylophilaceae from other closely related methanogenic archaea, easily leading to insufficient specificity, false positives, or missed detections, making it difficult to achieve precise targeted monitoring and quantitative analysis of this functional group.

[0004] The products encoded by functional genes directly participate in specific metabolic pathways, and their sequences may have higher distinguishability among different physiological groups. mtaBThe gene encodes methanol methyltransferase, a key functional gene in the methanogenesis pathway of Methanomethylophilaceae using methyl compounds. Therefore, we propose primers, kits, and PCR methods for detecting Methanomethylophilaceae in the cellar mud of strong-aroma baijiu, in order to alleviate or solve the aforementioned problems.

[0005] The information disclosed above in this background section is only for enhancing the understanding of the background section of this invention, and therefore may include prior art that is not known to those skilled in the art. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides primers, kits, and PCR methods for detecting Methanomethylophilaceae in the cellar mud of strong-aroma baijiu. These methods solve the problems of traditional culture methods failing to effectively detect the bacteria, existing molecular detection methods having poor specificity and high false positive rates, and the lack of precise quantitative detection methods for trace amounts of Methanomethylophilaceae in the complex matrix of cellar mud, thus hindering the resource exploration and functional analysis of this bacterium.

[0007] To achieve the above objectives, the present invention provides a primer pair for the specific identification of Methanomethylophilaceae. The primer pair is specific to the above-mentioned... mtaB Designed using conserved gene sequences, the primer pair includes upstream and downstream primers. This primer pair can specifically amplify a fragment of approximately 250 bp in length from the genomic DNA of Methanomethylophilaceae, while exhibiting no amplification activity against other common methanogenic archaea such as Methanopylocoridae and Methanopyrophagaceae, demonstrating good species specificity.

[0008] A qualitative detection method for Methanomethylophilaceae for non-diagnostic purposes, comprising the following steps: (a) Provide the genomic DNA of the sample to be tested; (b) Using the genomic DNA obtained in step (a) as a template, polymerase chain reaction (PCR) amplification was performed using the primer pairs described above; (c) If the amplification product of step (b) contains a DNA fragment of 250 bp, then the sample to be tested is determined to contain Methanomethylophilaceae.

[0009] A quantitative detection method for Methanomethylophilaceae in the cellar mud of strong-aroma baijiu includes the following steps: S1. Provide genomic DNA from pit mud samples; S2. Using the genomic DNA obtained in step S1 as a template, perform real-time quantitative PCR (qPCR) amplification using the above primer pairs, and record the Ct value; S3. Based on the Ct value obtained in step S2, and referring to the standard curve constructed using nucleic acid molecules containing the sequence shown in SEQ ID NO: 2, calculate the functional gene of Methanomethylophilaceae in the pit mud sample. mtaB The number of copies.

[0010] The PCR detection method provided by this invention includes a conventional PCR qualitative detection method and a qPCR quantitative detection method. The conventional PCR qualitative detection method is used to quickly screen whether a sample contains Methanomethylophilaceae, while the qPCR quantitative detection method is used to accurately determine the abundance of Methanomethylophilaceae in the sample.

[0011] Compared with the prior art, the beneficial effects of the present invention are: This invention is the first to target the methanol methyltransferase encoding gene unique to Methanomethylophilaceae. mtaB Specific primers were designed for this gene sequence, which is highly conserved in Methanomethylophilaceae but shows low homology or significant differences in other methanogenic archaea. Experimental validation showed that the provided primer pair amplified a single 250 bp target band only against Methanomethylophilaceae, while showing no non-specific amplification against various other families and genera of methanogenic archaea, such as Luminiformis muscarinae, Methanotropha martensii, and Methanotropha natans. This overcomes the cross-reactivity and false positive problems that may be caused by universal primers relying on 16S rRNA, achieving specific identification of the target bacterial group at the family level and ensuring the accuracy of the detection results.

[0012] This invention integrates real-time quantitative PCR technology, enabling precise quantification of trace amounts of Methanomethylophilaceae genomic DNA in pit mud samples. The established standard curve exhibits good linearity, meets amplification efficiency requirements, and has a low detection limit. This allows the method to effectively address situations where the total microbial population in pit mud is large, but the relative abundance of target bacterial groups may be low, achieving sensitive detection and accurate quantification of Methanomethylophilaceae and avoiding missed detections due to low abundance.

[0013] The entire detection process of this invention eliminates the need for isolation and purification of the difficult-to-cultivate Methanomethylophilaceae. Starting with the direct extraction of total DNA from the pit mud sample, qualitative or quantitative analysis can be completed within hours through PCR amplification and subsequent analysis. This shortens the detection cycle and improves work efficiency.

[0014] The technical solution of this invention is directly derived from the metagenomics of strong-aroma baijiu cellar mud. The experimental verification takes cellar mud samples and cellar mud enrichment liquid as the objects, takes into account the characteristics of cellar mud as a complex matrix, and after repeated verification with actual cellar mud samples, it is proven that it can effectively eliminate background interference, is stable, and can be applied to sample detection in real production environments.

[0015] The methylmethanophile (VadinCA11 sp. JN-1) used for primer verification in this invention has the accession number CICC25540, the accession date is June 10, 2026, and the accession center is the China Industrial Microbiological Culture Collection Center (CICC), located at Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing, postal code 100015. It belongs to the genus VadinCA11 of the Methanomethylophilaceae family. Other strains are commercially available strains, and their strain numbers are shown in Table 2.

[0016] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the phylogenetic tree constructed from the 16S rRNA gene sequence (SEQ ID NO: 1) of VadinCA11 sp.JN-1 and 29 highly homologous sequences in the NCBI database, as shown in this embodiment of the invention.

[0018] Figure 2 VadinCA11 sp.JN-1 of the present invention mtaB A schematic diagram of the phylogenetic tree constructed from the gene sequence (SEQ ID NO: 2) and 23 representative homologous sequences in the NCBI database.

[0019] Figure 3 This is an agarose gel electrophoresis image showing the PCR amplification of methanogenic archaea from different families and genera using the specific primer pairs (SEQ ID NO: 3 and SEQ ID NO: 4) designed in this invention. Wherein: M: Marker, 1: VadinCA11 sp. JN-1, 2: Luminidome methanogenic muscarinicella (…Methanomassiliicoccus luminyensis ), 3: Methanococcus martensii ( Methanosarcina mazei ), 4: Methanococcus marinei ( Methanococcus maripaludis ), 5: Bresbene methanogens ( Methanoculleus bourgensis ), 6: Black Sea methanogens ( Methanoculleus marisnigri ), CK:ddH2O.

[0020] Figure 4 This is an electrophoresis image used in this invention to verify the exclusivity of PCR amplification of various methanogenic archaea using primer pairs SEQ ID NO: 5 and SEQ ID NO: 6. Wherein: M: Marker, 1: VadinCA11 sp. JN-1, 2: Luminidome methanogenus (…). Methanomassiliicoccus luminyensis ), 3: Methanococcus martensii ( Methanosarcina mazei ), 4: Methanococcus marinei ( Methanococcus maripaludis ), 5: Bresbene methanogens ( Methanoculleus bourgensis ), 6: Black Sea methanogens ( Methanoculleus marisnigri ), CK:ddH2O.

[0021] Figure 5 This is an electrophoresis image used in this invention to verify the exclusivity of PCR amplification of various methanogenic archaea using primer pairs SEQ ID NO: 7 and SEQ ID NO: 8. Wherein: M: Marker, 1: VadinCA11 sp. JN-1, 2: Luminidome methanogenus (…). Methanomassiliicoccus luminyensis ), 3: Methanococcus martensii ( Methanosarcina mazei ), 4: Methanococcus marinei ( Methanococcus maripaludis ), 5: Bresbeckia orientalis ( ) Methanoculleus bourgensis ), 6: Black Sea methanogens ( Methanoculleus marisnigri ), CK:ddH2O.

[0022] Figure 6 This is an inclusion verification electrophoresis image of PCR amplification of Methanomethylophilaceae enrichment solutions from different sources using primer pairs SEQ ID NO: 3 and SEQ ID NO: 4. Wherein: M: Marker, 1: J-1, 2: J-2, 3: J-3, 4: J-4, 5: J-5, 6: J-6, 7: J-2 enrichment solution, 8: J-3 enrichment solution, 9: J-4 enrichment solution, 10: J-5 enrichment solution, 11: J-6 enrichment solution, CK: ddH2O.

[0023] Figure 7 For Methanomethylophilaceae mtaBStandard curve plot of gene copy number versus Ct value. Detailed Implementation

[0024] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. It should be noted that the drawings are schematic and not illustrated to scale. For clarity and convenience, the relative sizes and proportions of the parts shown in the drawings have been exaggerated or reduced in size. Any size is only illustrative and not limiting.

[0025] Example 1: Design, screening, and validation of Methanomethylophilaceae-specific primers

[0026] 1.1 Obtaining and analyzing target sequences Genomic sequence information of VadinCA11 sp. JN-1 was obtained from metagenomic assembly analysis of the J-1 mud sample. The 16S rRNA sequence of VadinCA11 sp. JN-1 (SEQ ID NO:1) was analyzed using NCBI BLAST, and nucleotide sequences with a sequence coverage greater than 90% and a sequence similarity greater than 75% were selected. After redundancy removal, 30 valid sequences were obtained, and a phylogenetic tree was constructed based on these sequences. The results are shown in the attached figure. Figure 1 As shown.

[0027] Comparative genomics analysis yielded a conserved nucleotide sequence (SEQ ID NO:2) encoding a protein in the VadinCA11 sp. JN-1 genome. This nucleotide sequence is presumed to function as a methanol methyltransferase (methanol: 5-hydroxy-benzimidazolyl-cobamide methyltransferase). mtaB The gene was retrieved. Homologous sequences were searched using the NCBI NT database, yielding 98 homologous sequences within the methanogenic archaea group. Only 5 sequences from the Methanomethylophilaceae family and 8 sequences from the Methanomassiliicoccaceae family had sequence similarity greater than 70% and sequence coverage greater than 90%. The remaining 69 sequences were from the Methanosarcinaceae family, with sequence similarity less than 70%. After deduplication of the 98 sequences from the methanogenic archaea genome, 23 representative sequences were obtained. A phylogenetic tree analysis was performed on this gene, along with the aforementioned 23 representative homologous sequences and sequences from the Methanobacteriaceae family (a total of 24 sequences). The results are attached. Figure 2 As shown.

[0028] The results showed that the nucleotide sequence shown in SEQ ID NO:2 is conserved and ubiquitous in Methanomethylophilaceae, with low homology in other methanogenic archaea, making it suitable as a target sequence for specific detection.

[0029] 1.2 Primer Design and Preliminary Screening For the nucleotide sequence shown in SEQ ID NO:2, specific primers were designed using Primer Premier 5 software. The primers were comprehensively evaluated using Oligo 7 software, including their Tm value, GC content, and dimer formation probability. Finally, three pairs of specific primers were selected, and the primer information is shown in Table 1.

[0030] Using the NCBI online tool Primer-BLAST, specificity simulations of three primer pairs were performed across the archaea genome, allowing for mismatches of no more than six base pairs per primer. The results showed that all three primer pairs specifically recognized the mtaB gene sequence of Methanomethylophilaceae, with no obvious non-specific binding sites.

[0031] Table 1: Primer Information for Methanomethylophilaceae

[0032] 1.3 Experimental verification of primers Primers were designed targeting the conserved sequence of the mtaB gene in *Methanomethylophilaceae* (as shown in SEQ ID NO: 2). Primers were designed using Primer Premier 5 and comprehensively evaluated using Oligo 7, ultimately selecting three pairs of specific primers, as shown in Table 1. Primer-BLAST was used to verify SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 7 and SEQ ID NO: 8 within the archaea genome, allowing for up to six base mismatches per primer. The results showed that all three primer pairs exhibited good specificity and could be used for subsequent physical validation.

[0033] To comprehensively evaluate the specificity of the three primer pairs, seven methanogenic archaea from different families were selected as experimental validation strains to verify primer exclusivity and optimize the best primer scheme. Among them, VadinCA11 sp. JN-1, with accession number CICC25540, was deposited on June 10, 2026, at the China Industrial Microbial Culture Collection Center (CICC), located at Building 6, No. 24, Jiuxianqiao Middle Road, Chaoyang District, Beijing, postal code 100015. It belongs to the genus VadinCA11 of the Methanomethylophilaceae family. Methanomassiliicoccus luminyensis , Methanosarcina mazei , Methanococcus maripaludis , Methanoculleus bourgensis MS2 and Methanoculleus marisnigri JR1 are publicly known strains, and their strain numbers are shown in Table 2. After extracting the enrichment solution or the genomic DNA of the strains, PCR amplification was performed using the primer sets shown in Table 1 to verify the specificity and effectiveness of the selected primers. The strain information is shown in Table 2.

[0034] Table 2 Information on the experimental strains for verification

[0035] Specifically, the PCR amplification system consisted of 50 μL, including 2 μL of DNA template, 2 μL x 2 of forward and reverse primers (10 μmol / L), 25 μL of 2×PCR Taqmix, and 19 μL of ddH2O. The amplification conditions were: 94 ℃ for 5 min; 94 ℃ for 30 s, 53 ℃ for 30 s, 72 ℃ for 30 s, for 30 cycles; and 72 ℃ for 10 min. The amplified products were subjected to gel electrophoresis, and the results are attached. Figure 3 - Appendix Figure 5 As shown.

[0036] The exclusion results for non-Methanomethylophilaceae are shown in Table 2. Figure 3 , Figure 4 , Figure 5 As shown, the primers shown in SEQ ID NO:3 and SEQ ID NO:4 did not show nonspecific amplification in non-Methanomethylophilaceae strains, indicating that the primers have good specificity;

[0037] This invention further uses six samples of cellar mud from different origins of strong-aroma baijiu and five corresponding enrichment cultures to verify primer amplification inclusiveness. The results are attached. Figure 6 As shown, all pit mud samples and enrichment cultures amplified a clear 250 bp target band without any extraneous bands.

[0038] The above results indicate that primers SEQ ID NO:3 and SEQ ID NO:4 have high amplification efficiency and good specificity on Methanomethylophilaceae strains, while other species not belonging to Methanomethylophilaceae cannot amplify the product.

[0039] Eleven PCR amplification products were sequenced to obtain nucleotide sequences of 250 bp in length. Comparison analysis showed that the sequence similarity was over 99%, and they were similar to those of Methanomethylophilaceae. mtaB The conserved gene sequences are highly homologous and show no significant homology with other families and genera of methanogenic archaea.

[0040] Weigh 0.5 g of cellar mud sample. Detailed information for samples 1-6 is shown in Table 3. Samples 7-11 are enrichment solutions, and CK is ddH2O. Genomic DNA was extracted from the cellar mud samples of strong-aroma baijiu cellars according to the Fast DNA SPIN Kit for soil instructions.

[0041] Table 3 Sample Information Table

[0042] PCR amplification was performed using the specific primer pairs (SEQ ID NO: 3 and SEQ ID NO: 4) screened in Example 1. The PCR amplification system was 50 μL, including 2 μL of DNA template, 2 μL × 2 of forward and reverse primers (10 μmol / L), 25 μL of 2×PCRTaqmix, and 19 μL of ddH2O. The amplification conditions were: 94 ℃ for 5 min; 94 ℃ for 30 s, 53 ℃ for 30 s, 72 ℃ for 30 s, for 30 cycles; 72 ℃ for 10 min. The amplification products were subjected to gel electrophoresis, and the results are as follows. Figure 6 As shown.

[0043] Sequencing and identification results TA cloning: using pMD TM The 19-T Vector Cloning Kit is used to ligate the recovered and purified PCR products to the T-vector. The ligation system is as follows: pMD TM0.4 μL of 19-T, 5 μL of Solution I, 60-100 ng of PCR product, and ddH2O to a final volume of 10 μL were mixed and incubated at 15°C for 30 min. The mixture was then transformed into Escherichia coli DH5α (Transgen) competent cells as follows: 100 μL of competent cells were added, along with 10 μL of ligation product. The mixture was incubated on ice for 5 min, then incubated in a 42°C water bath for 45 s, followed by an ice bath for 1 min. 500 μL of LB liquid medium was added, and the cells were incubated at 37°C for 1 h to recover. 40 μL of X-gal and 7 μL of IPTG were added to an LB agar plate containing Amp resistance. 100 μL of the recovered transformation product was spread onto the plate surface and incubated inverted at 37°C overnight.

[0044] Positive clone detection: Based on the blue-white screening principle, 20 positive clones were selected and sent to Qingke Biotechnology for sequencing. Comparative analysis of the 20 sequences showed that 16 sequences had a similarity greater than 99.2% to SEQ ID NO:1, and the remaining 4 sequences had a similarity greater than 99.6% to SEQ ID NO:2. The results indicate that the primer sets shown in SEQ ID NO:3 and SEQ ID NO:4 can specifically amplify Methanomethylophilaceae in the cellar mud of strong-aroma baijiu.

[0045] Based on the aforementioned primer pairs SEQ ID NO:3 and SEQ ID NO:4, which have been verified to have excellent specificity and inclusiveness, this invention further establishes a real-time quantitative PCR method for detecting Methanomethylophilaceae in the cellar mud of strong-aroma baijiu. Since this primer pair can only specifically amplify the mtaB gene of Methanomethylophilaceae, this qPCR method does not suffer from quantitative deviation problems caused by non-specific amplification.

[0046] The genomic DNA extraction was performed in the same manner as in Example 2.

[0047] In the real-time quantitative PCR detection, the qPCR reaction system consisted of: 25 μL total volume: 12.5 μL SYBR qPCR mix, 0.5 μL × 2 primers (10 μmol / L), 2 μL diluted DNA template, and 6.5 μL sterile ultrapure water. Amplification was performed using an ABI 7500 real-time quantitative PCR instrument. The qPCR amplification conditions were: 95℃ for 2 min, 95℃ for 10 s, 55℃ for 30 s, and 72℃ for 30 s, for a total of 40 cycles. The detection results are shown in Table 4.

[0048] Table 4. Methanomethylophilaceae detected in pit mud by qPCR method mtaB Gene copy number results table

[0049] Using pMD TM Using the 19-T Vector Cloning Kit, the recovered PCR product was ligated into the T-vector and transformed into Escherichia coli DH5α competent cells. 100 μL of the recovered transformation product was plated on an LB agar plate and incubated overnight at 37°C. Positive clones were picked and cultured, followed by plasmid extraction. Based on plasmid sequence analysis, the circularized plasmid was linearized using restriction endonucleases, and the template was serially diluted from 10... -1 Up to 10 -8 Real-time fluorescence quantification was performed, and a standard curve of qPCR amplification Ct value versus gene copy number was established. The formulas for template concentration and copy number are as follows: where CN (Copy Number) is the copy number, c is the genomic DNA concentration, and n is the genomic DNA fragment length.

[0050]

[0051] As attached Figure 7 As shown, the regression equation for the standard curve is y = -3.1383x + 68.424, where y represents the Ct value of qPCR amplification and x represents the gene copy number. The amplification efficiency of the standard curve is 107%, which is within the theoretical range of 90% to 110%, indicating good amplification efficiency. The correlation coefficient R of the standard curve is [value missing]. 2 =0.9998, indicating a good linear relationship between the Ct value of qPCR amplification and the gene copy number.

[0052] Set up blank, negative, and positive controls. The Ct values ​​for the blank and negative controls should be greater than 30, and the Ct value for the positive control should be less than 25. The Ct value of the sample to be tested should be within the range of the standard curve. If it is not within the range, the sample DNA concentration needs to be adjusted appropriately before retesting. For unknown samples that meet the above requirements, their Ct values ​​can be substituted into the standard curve, and the concentration of Methanomethylophilaceae can be obtained after equation conversion. mtaB Gene copy number.

[0053] In summary, the specific primers developed in this embodiment for identifying methylophilic methanogens based on gene sequences are specific at the Methanomethylophilaceae family level, yielding a 250 bp target product. Strains that produce the target band using PCR can be identified as Methanomethylophilaceae. Experiments have confirmed that this primer pair has good detection accuracy. The specificity of the primer pairs shown in SEQ ID NO:3 and SEQ ID NO:4 was verified within the archaea genome using the NCBI primer tool Primer-BLAST, allowing for no more than 6 base mismatches per primer. The primer pairs shown in SEQ ID NO:3 and SEQ ID NO:4 only yielded results for Methanomethylophilaceae in the NCBI database, indicating good specificity for this family.

[0054] The specificity of this qPCR method has been verified by routine PCR exclusion and sequencing of amplified products in Example 1. All qPCR amplified products were detected by agarose gel electrophoresis as a single 250 bp target band, and the melting curve showed a single sharp peak. There were no primer dimers or non-specific amplified products, which further proved the quantitative accuracy of this method.

[0055] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0056] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A primer pair for the specific detection of Methanomethylophilaceae, characterized in that, The primer pair consists of an upstream primer and a downstream primer, the nucleotide sequence of the upstream primer is shown in SEQ ID NO: 3, and the nucleotide sequence of the downstream primer is shown in SEQ ID NO:

4.

2. A kit for the specific detection of Methanomethylophilaceae, characterized in that, It includes the primer pair as described in claim 1.

3. A qualitative detection method for Methanomethylophilaceae for non-diagnostic purposes, characterized in that, Includes the following steps: (a) Provide the genomic DNA of the sample to be tested; (b) Using the genomic DNA obtained in step (a) as a template, polymerase chain reaction (PCR) amplification was performed using the primer pair described in claim 1; (c) If the amplification product of step (b) contains a DNA fragment of 250 bp, then the sample to be tested is determined to contain Methanomethylophilaceae.

4. A quantitative detection method for Methanomethylophilaceae in the cellar mud of strong-aroma baijiu, characterized in that, Includes the following steps: S1. Provide genomic DNA from pit mud samples; S2. Using the genomic DNA obtained in step S1 as a template, perform real-time quantitative PCR (qPCR) amplification using the primer pair described in claim 1, and record the Ct value; S3. Based on the Ct value obtained in step S2, and referring to the standard curve constructed using nucleic acid molecules containing the sequence shown in SEQ ID NO: 1, calculate the functional gene of Methanomethylophilaceae in the pit mud sample. mtaB The number of copies.

5. The use of the primer pair of claim 1 in the preparation of reagents or kits for detecting Methanomethylophilaceae.

6. The application of the primer pair of claim 2 or the kit of any one of claims 3-4 in any of the following non-diagnostic purposes: analysis of the microbial community of cellar mud during the brewing process of strong-aroma baijiu; quality assessment or functional monitoring of cellar mud for strong-aroma baijiu; screening or identification of Methanomethylophilaceae strains.

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