CN118086544A - Quantitative detection method for bacillus thallus in Daqu fermentation process - Google Patents

Abstract

The invention relates to a quantitative detection method of bacillus thallus in a Daqu fermentation process, and belongs to the field of fermented foods. The method takes a specific sequence in bacillus genome as a target detection fragment, adopts a bioinformatics method to design and synthesize a specific primer sequence covering the target fragment, combines a fluorescent real-time quantitative nucleic acid amplification technology, and quantitatively analyzes bacillus in a Daqu solid state fermentation system. Compared with other microorganism counting methods in the Daqu fermentation process, the method has the advantages of simplicity and convenience in operation, rapidness, high efficiency, strong specificity, high accuracy, economy, practicability and the like.

Description

Quantitative detection method for bacillus thallus in Daqu fermentation process

Technical Field

The invention relates to the technical field of traditional brewing, in particular to a quantitative detection method of bacillus thallus in a Daqu fermentation process.

Background

The medium-high temperature Daqu is usually prepared by using wheat and adding auxiliary materials such as peas according to a certain proportion, wherein the highest temperature can reach 50-60 ℃ in the process of fermenting the yeast, and the white spirit brewed by using the distiller's yeast has strong aroma, so the distiller's yeast is also called as the strong aroma type Daqu. The middle-high temperature Daqu fermentation adopts an open environment for spontaneous fermentation, and the yeast blocks are in direct contact with the environment in the fermentation process, so that various microorganisms are rich in content, a complex solid fermentation system is formed by combining the open fermentation conditions, relatively harsh environmental conditions such as high temperature, high humidity and the like can be formed in the Daqu fermentation process, and a unique microorganism structure is formed in the fermentation process. In the fermentation process of Daqu, the community structure of bacteria and fungi is changed remarkably in the fermentation process, wherein the bacterial community structure is greatly influenced by environmental pressure, and the fungus community structure is relatively stable. Among bacteria, bacillus is a common dominant bacterium in the fermentation process of Daqu.

The bacillus is an important functional bacterium in the fermentation process of the Daqu, and the metabolic activity of the bacillus is beneficial to improving physical and chemical indexes of the Daqu, strengthening the flavor of the Daqu and adjusting the microbial structure in the fermentation process of the Daqu. Bacillus licheniformis can produce various antibacterial substances (such as bacitracin, antibacterial proteins, etc.) and nutrients (such as vitamins, growth promoting factors, etc.), and can regulate microbial structures of flora. In addition, bacillus licheniformis can also produce short chain fatty acids (short CHAIN FATTY ACID, SCFA), lowering environmental pH. Lysine bacillus exists widely in natural environment, such as root systems of crops like capsicum, and because of strong stress resistance, lysine bacillus always keeps high abundance in the whole fermentation process of Daqu, and is considered as one of white wine aroma-enhancing strains.

In recent years, with the development of microbiology, targeted amplicon sequencing (prokaryote of 16SrRNA gene), 18S rRNA of eukaryote and internal transcription spacer [ ITS ] gene have opened a new era of microbiological study, and knowledge of microbial composition and function of Daqu has been improved through sequence clustering, OTU partitioning and comparison of databases (bacterial and archaea 16S rRNA database, fungus 18S rRNA database, fungus ITS database and functional gene database). However, no doubt, the use of histology requires more capital support and the ability to analyze and mine large-scale data sets, and such methods all suffer from the problem of long sequencing cycles and inability to obtain results quickly.

The invention aims at overcoming the defects of the prior art, and provides a method for quantitatively detecting microorganisms in the traditional brewing process, in particular to a method for quantitatively detecting bacillus in the mixed microorganism solid state fermentation process. The method has the characteristics of accurate result, high speed, high efficiency and high sensitivity, and solves the problems that the traditional counting method is long in time consumption and high in operation intensity, and target microorganisms cannot be specifically quantified.

Disclosure of Invention

In order to solve the technical problems, the invention provides a quantitative detection method of bacillus thallus in the fermentation process of Daqu. The invention uses the specific sequence in bacillus genome as target detection fragment, adopts bioinformatics method to design and synthesize specific primer sequence covering target fragment, combines fluorescent real-time quantitative nucleic acid amplification technology, and quantitatively analyzes bacillus in Daqu solid state fermentation system. The method has the advantages of simplicity and convenience in operation, rapidness, high efficiency, strong specificity, high accuracy, economy, practicability and the like.

The invention is realized by the following technical scheme:

the first object of the invention is to provide a specific primer for quantitative detection of bacillus thallus in the fermentation process of Daqu, wherein the nucleotide sequence of the specific primer is as follows:

Specific primer upstream primer SC4 of bacillus licheniformis (the sequence is shown as SEQ ID NO. 1)

And the downstream primer SCA4 (sequence shown as SEQ ID NO. 2) is:

the upstream primer SC4:5'-TTGGCTCGAAGCGTAATTGA-3';

Downstream primer SCA4:5'-CTTGGGAAGAAGCGGTATCC-3';

The specific primers of the lysine bacillus, namely an upstream primer LB2 (the sequence of which is shown as SEQ ID NO. 3) and a downstream primer LBA2 (the sequence of which is shown as SEQ ID NO. 4) are as follows:

upstream primer LB2:5'-GGGAATGTTGGTGAAGCACT-3';

downstream primer LBA2:5'-ACCGTAAGTTGTGCAGATTCC-3'.

The second purpose of the invention is to provide the application of the specific primer in quantitative detection of bacillus thallus in the fermentation process of Daqu.

The invention provides a quantitative detection method of bacillus thallus in the fermentation process of Daqu, which comprises the following steps:

(1) Taking a thallus sample in the fermentation process of Daqu, re-suspending, removing impurities, and cleaning thallus to obtain a thallus suspension; precipitating thalli in the obtained bacterial suspension, and extracting sample DNA;

(2) Using bacillus as a standard substance, carrying out gradient dilution, plating counting, bacterial precipitation and DNA extraction;

(3) Respectively taking DNA prepared by a standard substance and a sample as templates, and adopting a specific primer to carry out fluorescent quantitative PCR reaction;

(4) And (3) fitting and calculating the linear relation between the bacillus thallus count and the Ct value, establishing a standard curve and carrying out sample analysis.

In one embodiment of the present invention, in the step (1), the method for removing impurities is: the solid residue was removed by filtration with gauze.

In one embodiment of the present invention, in the step (1), the method for cell precipitation comprises: centrifuging the bacterial suspension at room temperature of 3000r/min-8000r/min for 3min-5min, and collecting the bacterial cells.

In one embodiment of the present invention, in step (2), the bacillus is bacillus licheniformis and/or bacillus lysine.

In one embodiment of the invention, in step (3), the nucleotide sequence of the specific primer is as follows:

Specific primers for bacillus licheniformis:

the upstream primer SC4:5'-TTGGCTCGAAGCGTAATTGA-3';

Downstream primer SCA4:5'-CTTGGGAAGAAGCGGTATCC-3';

specific primers for lysine bacillus:

upstream primer LB2:5'-GGGAATGTTGGTGAAGCACT-3';

downstream primer LBA2:5'-ACCGTAAGTTGTGCAGATTCC-3'.

In one embodiment of the present invention, in the step (3), the reaction system of the fluorescent quantitative PCR reaction is: 2X SYBRqPCR mix 12.5.5. Mu.L, 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 1. Mu.L to 2.5. Mu.L (preferably 2.5. Mu.L) of total genomic DNA of the sample to be detected, and 9. Mu.L to 10.5. Mu.L (preferably 9.0. Mu.L) of ddH ₂ O, totaling 25.0. Mu.L.

In one embodiment of the present invention, in step (3), the reaction procedure of the fluorescent quantitative PCR reaction is: pre-denaturation at 95℃for 3min; denaturation at 95℃for 3s-5s (preferably 5 s), annealing at 60℃for 20s-30s (preferably 20 s), fluorescence signal acquisition, 40 cycles were set; melting curve analysis: denaturation at 95℃for 15s, annealing at 60℃for 1min, extension at 95℃for 15s, annealing at 60℃for 15s.

In one embodiment of the present invention, in the step (4), the method for establishing a standard curve is:

Extracting the concentration of genome DNA according to the target detection bacteria, converting the concentration into gene copy number, and fitting the calculated gene copy number with the lithographic colony count to obtain a linear equation; and performing five-fold gradient dilution on the obtained genome standard substance to serve as a standard template solution for fluorescence quantitative PCR reaction, taking the logarithm of the concentration of the standard substance as an ordinate, taking the Ct value of the fluorescence quantitative PCR, namely the number of cycles undergone when the fluorescence signal in each reaction tube reaches a set threshold value as an abscissa, and drawing a standard curve.

Compared with the prior art, the technical scheme of the invention has the following advantages:

Since the Daqu fermentation adopts open spontaneous fermentation, a plurality of bacteria are planted in the fermentation process, and due to the variety of bacillus, in a mixed system, different bacillus species are difficult to distinguish and count by a conventional counting method, and quantitative detection means are required to be established for specific bacillus strains because part of key strains need to be ascertained on Daqu fermentation.

The invention provides a quantitative detection method of bacillus thallus in a Daqu fermentation process. The fluorescent real-time quantitative PCR can rapidly and accurately quantify the concentration of nucleic acid in a sample, so that the fluorescent real-time quantitative PCR can be used for classifying and quantitatively researching microorganisms in a brewing system. The invention relates to a quantitative detection method for the quantity of specific microbial cells in the solid state fermentation process of Daqu, which has the advantages of simple operation, high speed, high efficiency, economy, practicability and the like compared with the microbial counting methods in the fermentation process of other Daqus.

The bacillus licheniformis and lysine bacillus specific primers provided by the invention can simultaneously quantify bacillus in a complex microorganism system through specific sequences, eliminate interference of other strains to bacterial count outside a target strain, improve working efficiency, and have the advantages of strong specificity and high sensitivity.

Drawings

In order that the invention may be more readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings, in which

FIG. 1 shows the qPCR amplification curve of Bacillus licheniformis in the present invention;

FIG. 2 shows the qPCR melting curve of Bacillus licheniformis in the present invention;

FIG. 3 is a qPCR quantitative curve of Bacillus licheniformis in the present invention;

FIG. 4 shows qPCR amplification curves of B.lysine in the present invention;

FIG. 5 shows qPCR melting curves of B.lysine in the present invention;

FIG. 6 is a qPCR quantitative curve of the lysine bacillus in the present invention;

FIG. 7 is an amplification curve of a comparative example in which the primer specificity is not strong in the present invention;

FIG. 8 is a melting curve of a comparative example in which the primer specificity is not strong in the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.

The invention provides a method for simultaneously and quantitatively detecting the quantity of bacillus licheniformis and lysine bacillus in a mixed microorganism fermentation system, which comprises the following steps:

(1) Sample pretreatment: 1g-5g of sample to be detected is taken, PBS buffer solution is added for resuspension, solid residues are removed through gauze filtration, and supernatant is taken to obtain bacterial suspension;

(2) Preparing pure bacterial liquid of target detection bacteria, taking a proper amount of bacterial suspension, adding sterile water for gradient dilution to 10 ^-5、10^-6、10^-7 three dilution gradients, coating three LB plates on each gradient, and counting bacterial colonies;

(3) Centrifuging the bacterial suspension prepared in the step (1) for 3-5 min at the room temperature of 3000-8000 r/min to collect bacterial cells;

(4) Sample DNA extraction: extracting DNA from the thalli obtained in the step (3);

(5) The quantity of the thalli of the sample to be detected is determined by fluorescent quantitative PCR, and the method is concretely as follows:

① Establishing a standard curve: extracting the concentration of genome DNA according to the target detection bacteria, converting the concentration into gene copy number, and fitting the calculated gene copy number with the lithographic colony count to obtain a linear equation; performing five-fold gradient dilution on the obtained genome standard substance to serve as a standard template solution for fluorescence quantitative PCR reaction, taking the logarithm of the concentration of the standard substance as an ordinate, taking the Ct value of the fluorescence quantitative PCR, namely the cycle number undergone when the fluorescence signal in each reaction tube reaches a set threshold value as an abscissa, and drawing a standard curve;

② Actual sample quantification: and (3) taking the DNA sample obtained in the step (4) as a template, carrying out fluorescent quantitative PCR reaction by adopting the specific primer designed in the step ①, and comparing the obtained Ct value with the standard curve obtained in the step ① to determine the bacterial count.

Preferably, the specific primers are respectively:

① Specific primers for detection of bacillus licheniformis (Bacillus licheniformis):

The upstream primer SC4:5'-TTGGCTCGAAGCGTAATTGA-3' A

Downstream primer SCA4:5'-CTTGGGAAGAAGCGGTATCC-3';

② Specific primers for detection of bacillus lysine (Lysinibacillus capsici):

upstream primer LB2:5'-GGGAATGTTGGTGAAGCACT-3' A

Downstream primer LBA2:5'-ACCGTAAGTTGTGCAGATTCC-3';

The amplification reaction system is as follows: 2X SYBRqPCR mix 12.5.5. Mu.L, 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 2.5. Mu.L of total genomic DNA of the sample to be detected, and 25.0. Mu.L in total of ddH ₂ O9.0. Mu.L.

The amplification reaction program is as follows: pre-denaturation at 95℃for 3min; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 20s, collecting fluorescent signals, and setting 40 cycles; melting curve analysis: denaturation at 95℃for 15s, annealing at 60℃for 1min, extension at 95℃for 15s, annealing at 60℃for 15s.

Example 1

The embodiment provides a method for quantitatively detecting bacillus licheniformis in the solid state fermentation process of Daqu, which comprises the following specific steps:

(1) Establishment of a Standard Curve

Experimental strains: bacillus licheniformis W22 (the laboratory screens and saves from Daqu, NCBI database strain number NZ_CP 014842.1)

Bacillus licheniformis was cultured overnight in LB medium to give an OD ₆₀₀ of 1, and appropriate amount of bacterial liquid was diluted to 10 ^-5、10^-6、10^-7 gradients, each of which was plated with three LB plates and colonies were counted.

Extracting genome of bacillus licheniformis with OD ₆₀₀ value of 1 as a standard template, and carrying out gradient dilution to prepare qPCR reaction systems respectively.

The amplification reaction system is as follows: 2X SYBRqPCR mix 12.5.5. Mu.L, 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 2.5. Mu.L of total genomic DNA of the sample to be detected, and 25.0. Mu.L in total of ddH ₂ O9.0. Mu.L.

The upstream primer SC4:5'-TTGGCTCGAAGCGTAATTGA-3';

Downstream primer SCA4:5'-CTTGGGAAGAAGCGGTATCC-3';

The amplification reaction procedure was: pre-denaturation at 95℃for 3min; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 20s, collecting fluorescent signals, and setting 40 cycles; melting curve analysis: denaturation at 95℃for 15s, annealing at 60℃for 1min, extension at 95℃for 15s, annealing at 60℃for 15s.

After the amplification reaction, a qPCR amplification curve (see FIG. 1) was obtained, while a melting curve (see FIG. 2) was added. As can be seen from FIG. 1, the dilution factor of the standard DNA is suitable; as can be seen from FIG. 2, the qPCR product was single, and non-specific amplification and abnormal amplification could be excluded.

Establishing a standard curve (see figure 3) between the Cq value (X) of the standard DNA and the logarithmic value (Y) of the corresponding bacterial count, and performing regression analysis to obtain a linear regression equation: y= -3.7783x+45.5103, r2=0.9997. The concentration of bacillus licheniformis was then determined by calculating the logarithm of 10.

(2) Taking 1g-5g of yeast block sample in the fermentation process of Daqu, adding PBS buffer solution for resuspension, filtering with gauze to remove solid residues, and taking supernatant to obtain bacterial suspension.

Centrifuging the prepared bacterial suspension at room temperature of 3000r/min-8000r/min for 3min-5min to collect bacterial cells;

Sample DNA extraction: extracting DNA from the obtained thalli;

And determining the number of the thalli of the sample to be detected by fluorescent quantitative PCR, wherein the reaction procedure is the same as that described above.

After the amplification reaction, the Cq value of the solution is 19.93, and the solution is substituted into a linear regression equation to obtain Y= 6.7703, and the number of bacillus licheniformis in each gram of sample is 10 ^6.7703.

Example 2

The embodiment provides a method for quantitatively detecting lysine bacillus in the solid state fermentation process of Daqu, which comprises the following specific steps:

(1) Establishment of a Standard Curve

Experimental strains: lysine bacillus X32 (the laboratory screens and saves from Daqu, NCBI database with the strain number NZ_CP 084108.1)

The lysine bacillus is cultured in LB culture medium overnight, when the OD ₆₀₀ is 1, a proper amount of bacterial liquid is taken and respectively diluted to 10 ^-5、10^-6、10^-7 gradients, three LB plates are coated on each gradient, and colony counting is carried out.

Extracting genome of lysine bacillus with OD ₆₀₀ value of 1 as standard template, and gradient diluting to prepare qPCR reaction system.

The amplification reaction system is as follows: 2X SYBRqPCR mix 12.5.5. Mu.L, 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 2.5. Mu.L of total genomic DNA of the sample to be detected, and 25.0. Mu.L in total of ddH2O 9.0. Mu.L.

Upstream primer LB2:5'-GGGAATGTTGGTGAAGCACT-3';

Downstream primer LBA2:5'-ACCGTAAGTTGTGCAGATTCC-3';

The amplification reaction procedure was: pre-denaturation at 95℃for 3min; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 20s, collecting fluorescent signals, and setting 40 cycles; melting curve analysis: denaturation at 95℃for 15s, annealing at 60℃for 1min, extension at 95℃for 15s, annealing at 60℃for 15s.

After the amplification reaction, qPCR amplification curves were obtained (see FIG. 4), while melting curves were added (see FIG. 5). As can be seen from fig. 4, the dilution factor of the standard DNA is suitable; as can be seen from FIG. 5, the qPCR product was single, and non-specific amplification and abnormal amplification could be excluded.

Establishing a standard curve (see fig. 6) between the Cq value (X) of the standard DNA and the logarithmic value (Y) of the corresponding bacterial count, and performing regression analysis to obtain a linear regression equation: y= -3.5849x+37.2289, r2=0.9998. The log of 10 is then calculated to determine the concentration of lysine bacillus.

(2) Taking 1g-5g of yeast block sample in the fermentation process of Daqu, adding PBS buffer solution for resuspension, filtering with gauze to remove solid residues, and taking supernatant to obtain bacterial suspension.

Centrifuging the prepared bacterial suspension at room temperature of 3000r/min-8000r/min for 3min-5min to collect bacterial cells;

Sample DNA extraction: extracting DNA from the obtained thalli;

And determining the number of the thalli of the sample to be detected by fluorescent quantitative PCR, wherein the reaction procedure is the same as that described above.

After the amplification reaction, the Cq value of the measured solution was 17.78, and was substituted into a linear regression equation to obtain Y= 5.4252, and the number of lysine bacillus in each gram of sample was 10 ^5.4252.

Comparative example

Experimental strains: bacillus licheniformis W22 (strain number NZ_CP014842.1 in NCBI database was selected from Daqu and stored in this laboratory).

Bacillus licheniformis is cultured overnight in LB culture medium, and genome of Bacillus licheniformis with OD ₆₀₀ of 1 is extracted as standard template, and the template is gradient diluted to prepare qPCR reaction system.

The amplification reaction system is as follows: 2X SYBRqPCR mix 12.5.5. Mu.L, 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 2.5. Mu.L of total genomic DNA of the sample to be detected, and 25.0. Mu.L in total of ddH ₂ O9.0. Mu.L.

The upstream primer SC4 (the sequence is shown as SEQ ID NO. 5): 5'-GTTAATCCGCTTTGCGTCA-3' A

Downstream primer SCA4 (sequence shown as SEQ ID NO. 6): 5'-GCTCTCTTTAAGCGCGAATC-3';

The amplification reaction procedure was: pre-denaturation at 95℃for 3min; denaturation at 95 ℃ for 5s, annealing at 60 ℃ for 20s, collecting fluorescent signals, and setting 40 cycles; melting curve analysis: denaturation at 95℃for 15s, annealing at 60℃for 1min, extension at 95℃for 15s, annealing at 60℃for 15s.

After the amplification reaction, qPCR amplification curves were obtained (see FIG. 7), while melting curves were added (see FIG. 8). The amplification curve shows that the Cq value is larger, the melting curve is not unimodal, and the melting curve is unstable.

In conclusion, it can be seen that the primer used has weak specificity, so that the peak value of the product fragment is not single, and the product fragment is particularly reflected in that the melting curve is not unimodal and has fluctuation and instability, and the Cq value is not stable enough and has no regularity and is more than 38.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.

Claims (10)

1. A specific primer for quantitatively detecting bacillus thallus in the fermentation process of Daqu, which is characterized by comprising the following nucleotide sequences:

Specific primers for bacillus licheniformis:

the upstream primer SC4:5'-TTGGCTCGAAGCGTAATTGA-3';

Downstream primer SCA4:5'-CTTGGGAAGAAGCGGTATCC-3';

specific primers for lysine bacillus:

upstream primer LB2:5'-GGGAATGTTGGTGAAGCACT-3';

downstream primer LBA2:5'-ACCGTAAGTTGTGCAGATTCC-3'.

2. The use of the specific primer of claim 1 for quantitatively detecting bacillus thallus in the fermentation process of Daqu.

3. The quantitative detection method of bacillus thallus in the fermentation process of Daqu is characterized by comprising the following steps:

(1) Taking a thallus sample in the fermentation process of Daqu, re-suspending, removing impurities, and cleaning thallus to obtain a thallus suspension; precipitating thalli in the obtained bacterial suspension, and extracting sample DNA;

(2) Using bacillus as a standard substance, carrying out gradient dilution, plating counting, bacterial precipitation and DNA extraction;

(3) Respectively taking DNA prepared by a standard substance and a sample as templates, and adopting a specific primer to carry out fluorescent quantitative PCR reaction;

(4) And (3) fitting and calculating the linear relation between the bacillus thallus count and the Ct value, establishing a standard curve and carrying out sample analysis.

4. A quantitative determination method according to claim 3, wherein in step (1), the method for removing impurities is: the solid residue was removed by filtration with gauze.

5. The quantitative determination method according to claim 3, wherein in the step (1), the method of bacterial cell precipitation is as follows: centrifuging the bacterial suspension at room temperature of 3000r/min-8000r/min for 3min-5min, and collecting the bacterial cells.

6. A quantitative detection method according to claim 3, wherein in step (2), the bacillus is bacillus licheniformis and/or bacillus lysine.

7. The quantitative detection method according to claim 3, wherein in step (3), the nucleotide sequence of the specific primer is as follows:

Specific primers for bacillus licheniformis:

the upstream primer SC4:5'-TTGGCTCGAAGCGTAATTGA-3';

Downstream primer SCA4:5'-CTTGGGAAGAAGCGGTATCC-3';

specific primers for lysine bacillus:

upstream primer LB2:5'-GGGAATGTTGGTGAAGCACT-3';

downstream primer LBA2:5'-ACCGTAAGTTGTGCAGATTCC-3'.

8. The quantitative detection method according to claim 3, wherein in the step (3), the reaction system of the fluorescent quantitative PCR reaction is: 2X SYBRqPCR mix 12.5.5. Mu.L, 0.5. Mu.L of upstream primer, 0.5. Mu.L of downstream primer, 1. Mu.L-2.5. Mu.L of total genomic DNA of the sample to be detected, and 25.0. Mu.L in total of ddH ₂ O9. Mu.L-10.5. Mu.L.

9. The quantitative detection method according to claim 3, wherein in the step (3), the reaction procedure of the fluorescent quantitative PCR reaction is: pre-denaturation at 95℃for 3min; denaturation at 95 ℃ for 3s-5s, annealing at 60 ℃ for 20s-30s, collecting fluorescent signals, and setting 40 cycles; melting curve analysis: denaturation at 95℃for 15s, annealing at 60℃for 1min, extension at 95℃for 15s, annealing at 60℃for 15s.

10. The quantitative determination method according to claim 3, wherein in the step (4), the method for establishing a standard curve is as follows:

Extracting the concentration of genome DNA according to the target detection bacteria, converting the concentration into gene copy number, and fitting the calculated gene copy number with the lithographic colony count to obtain a linear equation; and performing five-fold gradient dilution on the obtained genome standard substance to serve as a standard template solution for fluorescence quantitative PCR reaction, taking the logarithm of the concentration of the standard substance as an ordinate, taking the Ct value of the fluorescence quantitative PCR, namely the number of cycles undergone when the fluorescence signal in each reaction tube reaches a set threshold value as an abscissa, and drawing a standard curve.