Quantitative detection method of caproic acid bacteria

Abstract

The invention belongs to the field of microorganism and molecule detection, and particularly relates to a quantitative detection method of caproic acid bacteria. Aiming at the problems of long detection time, tedious operation steps, low flux, high requirements on strict anaerobic conditions, easy infection of infectious microbes and the like of a traditional detection method, the invention provides a novel caproic acid bacteria detection method, a qPCR specific primer pair is designed based on recA genes of to-be-detected caproic acid bacteria, and the caproic acid bacteria are detected by using the qPCR specific primer pair. According to the primer pair and the method provided by the invention, the identification efficiency of the caproic acid bacteria can be effectively improved, a caproic acid bacteria screening method is optimized, and a quicker detection method is provided for related research of the caproic acid bacteria.

Description

Technical Field

[0001] This invention belongs to the field of microbiology and molecular detection, and specifically relates to a quantitative detection method for caproic acid bacteria. Background Technology

[0002] Hexanoic acid bacteria are key functional microorganisms in the brewing of strong-aroma baijiu. The hexanoic acid produced by their metabolism reacts with ethanol to form ethyl hexanoate, a major component of the main aroma of strong-aroma baijiu. The abundance of hexanoic acid bacteria in the fermentation pit mud is closely related to the quality of the pit mud and the flavor and quality of the baijiu. Therefore, real-time monitoring of changes in the abundance of hexanoic acid bacteria is crucial for process control, especially when the system faces shocks or operational instability. Timely understanding of the microbial community dynamics helps maintain operational stability and ensure processing efficiency.

[0003] However, due to the presence of various microorganisms in the complex environment of baijiu brewing, it is impossible to directly identify and quantify caproic acid bacteria using conventional plate counting methods. This severely restricts the effective monitoring of microbial community changes in fermentation pits or wastewater systems, thus affecting the accuracy and effectiveness of actual control. Furthermore, since most caproic acid bacteria are obligate anaerobes, they are difficult to culture on conventional agar plates, grow slowly, and exhibit poor reproducibility, further hindering the quantitative detection of caproic acid bacteria.

[0004] Given the limitations of the plate method, such as long detection time, cumbersome operation steps, low throughput, high requirements for strict anaerobic conditions, and susceptibility to contamination by other microorganisms, the development of rapid and accurate quantitative detection technology for caproic acid bacteria is particularly urgent. This type of technology can significantly shorten detection time, improve monitoring efficiency, and meet the need for real-time monitoring of microbial dynamics during brewing fermentation and wastewater treatment, providing a basis for precise regulation. For example, in cellar mud maintenance or artificial cellar mud construction, accurately obtaining the number of caproic acid bacteria can guide the strategy of adding microbial solution and enhance the function of the cellar mud; in wastewater treatment, treatment processes and parameters can be optimized based on bacterial concentration data to improve caproic acid synthesis efficiency and reduce operating costs; in microbial agent production, culture conditions and process parameters can be optimized based on caproic acid bacteria concentration data to obtain higher concentration microbial agent products.

[0005] Among various rapid detection methods, qPCR technology is particularly suitable for detecting difficult-to-culture microorganisms such as Hexanoic acid bacteria due to its advantages such as real-time quantification, high sensitivity, strong specificity, rapid detection (<2 hours), and good reproducibility. Commonly used target genes for this technology include conserved 16S rRNA genes, species-specific genes, and functional genes. However, the copy number of the 16S rRNA gene varies in the genomes of different bacteria, typically between 3 and 6, which leads to severe distortion in the quantitative results and fails to reflect the true cell count. Furthermore, the high conservation of the 16S rRNA gene makes it difficult to design specific primers that can distinguish closely related species or strains.

[0006] Therefore, if a new, accurate, and rapid detection method for caproic acid bacteria can be provided, it will have significant application prospects in fields such as liquor brewing. Summary of the Invention

[0007] The purpose of this invention is to provide a quantitative detection method for caproic acid bacteria.

[0008] To achieve the above-mentioned objective, the technical solution adopted by the present invention is: a set of primer pairs, wherein the primer pairs include an upstream primer P4f, the sequence of which is shown in SEQ ID NO: 8, and a downstream primer P4r, the sequence of which is shown in SEQ ID NO: 9.

[0009] Accordingly, detection reagents, test strips, or kits containing the primer pairs.

[0010] Accordingly, the primer pair or the detection reagent, test strip or kit is used in the detection of caproic acid bacteria.

[0011] Accordingly, a method for detecting caproic acid bacteria involves designing a qPCR-specific primer pair based on the recA gene of the target caproic acid bacteria, and using the qPCR-specific primer pair to detect the bacteria. The primer pair includes an upstream primer P4f, the sequence of which is shown in SEQ ID NO: 8, and a downstream primer P4r, the sequence of which is shown in SEQ ID NO: 9. The method includes the following steps:

[0012] (1) Design qPCR specific primer pairs based on the recA gene of the bacteria to be tested;

[0013] (2) Extract total DNA from the sample to be tested and perform qPCR amplification using the primer pair; if a band appears, the sample is considered to contain the target hexanoic acid bacteria; if no band appears, the sample is considered not to contain the target hexanoic acid bacteria. The 10 μL qPCR amplification reaction system includes: 2 μL of 2×SsoAdvanced Universal SYBR Green Supermix, 0.2 μL each of forward and reverse primers, and 1 μL of standard plasmid template. 3.6 μL. The qPCR amplification reaction program included: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, for 40 cycles.

[0014] (3) Constructing the standard curve:

[0015] (3-1) Plasmid concentration determination and copy number calculation: Determine the concentration of the standard plasmid and calculate the copy number; the formula for calculating the copy number is: copy number = The copy number is measured in copies / μL, and the total plasmid length is measured in bp.

[0016] (3-2) Standard curve plotting: Using the logarithm of the standard plasmid copy number as the x-axis and the Ct value as the y-axis, a standard curve is generated, and the regression equation and correlation coefficient are calculated. ;

[0017] (4) Quantitative detection of caproic acid bacteria: Substitute the Ct value of the sample to be tested into the regression equation of the standard curve to calculate the copy number of gene recA in the sample to be tested, thereby obtaining the cell content of caproic acid bacteria to be tested.

[0018] This invention offers the following advantages: Compared to conserved 16S rRNA genes, housekeeping genes such as rpoB, recA, and tuf are single copies in the vast majority of bacterial genomes. Furthermore, because housekeeping genes exhibit higher sequence variability than 16S rRNA genes, it is easier to design highly specific primers targeting genus, species, or even strains. This higher specificity reduces the risk of non-specific amplification in complex microbial communities. Detection of specific or functional genes enables precise identification at the species or strain level, effectively avoiding cross-reactivity and overcoming the limitations of traditional methods and 16S rRNA-based qPCR in quantification.

[0019] Based on the recA gene, this invention specifically designs primer pair P4 and further provides a quantitative PCR method using fluorescent dyes (qPCR), which can effectively improve the identification efficiency of caproic acid bacteria, optimize the screening method of caproic acid bacteria, and provide a faster detection method for caproic acid bacteria-related research. Attached Figure Description

[0020] Figure 1 This is a colony morphology diagram of the caproic acid bacteria to be detected in this invention;

[0021] Figure 2 Electrophoresis diagrams for detecting different objects using different primer pairs;

[0022] Figure 3 Electrophoresis image of recA recombinant plasmid;

[0023] Figure 4 The melting curve of SYBR Green I fluorescent dye qPCR method;

[0024] Figure 5 This is the standard amplification curve for the SYBR Green I fluorescent dye qPCR method;

[0025] Figure 6 This is the standard curve for the SYBR Green I fluorescent dye qPCR method. Detailed Implementation

[0026] This invention provides a rapid quantitative detection method for caproic acid bacteria. The method is based on the specific sequence recombinase RecA (GenBank: ARP49720.1, sequence shown in SEQ ID NO: 1) of caproic acid bacteria commonly found in strong-aroma baijiu fermentation pits (such as *Lactobacillus lactis* and *Rumen caproic acid bacteria*), and designs qPCR primer pairs. These primer pairs are then used for PCR amplification, identification, and quantitative detection. This invention is particularly suitable for the quantitative detection of *Rumen caproic acid bacteria* (GDMCC No. 60133) and *Lactobacillus lactis* (GDMCC 1.1627). The primer pairs only produce clear band amplification against these caproic acid bacteria. The specific detection method includes the following steps:

[0027] 1. Design specific primer pairs based on the specific gene recA.

[0028] 2. Construct a standard curve.

[0029] 2-1. Plasmid Concentration Determination and Copy Number Calculation: The concentration (ng / μL) of the standard plasmid was determined using a UV spectrophotometer, and the copy number was calculated using the formula:

[0030] Copy number (copies / μL) = / (Total plasmid length (bp) × 660 g / mol / bp). Total plasmid length (WG94303_pUC57, 3874 bp) = Vector length (pUC57, 2710 bp) + Fragment length (RecA gene fragment, 1164 bp).

[0031] 2-2. Serial dilution: Dilute the standard plasmid 10-fold serially with ddH2O (e.g., 10... 7 10 6 10 5 10 4 10 3 (Copies / μL), store temporarily at 4℃ (avoid repeated freeze-thaw cycles).

[0032] 2-3. qPCR Amplification and Standard Curve Construction: The logarithm (lg) of the copy number of the recombinant plasmid containing the recA gene is plotted on the x-axis, and the Ct value on the y-axis. qPCR reaction system (10 μL): 2 × Sso Advanced Universal SYBR Green Supermix 2 μL, forward and reverse primers (10 μmol / L) 0.2 μL each, plasmid template 1 μL. 3.6 μL. Reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 40 cycles. A standard curve was generated using the qPCR instrument's built-in software, and the regression equation (y=ax+b) and correlation coefficient were calculated. , ≥0.99).

[0033] 3. Detection of caproic acid bacteria

[0034] 3-1. Qualitative Detection: Extract total DNA from the sample to be tested (e.g., a bacterial genome assay kit can be used for liquid samples such as fermentation broth, and a soil DNA extraction kit can be used for solid samples such as pit mud), and perform qPCR amplification and detection together with the standard plasmid (a plasmid containing the recA gene). Amplification conditions are the same as in steps 2-3. Only hexanoic acid bacteria, especially rumen bacteria and Lactobacillus, will show bands.

[0035] 3-2. Quantitative calculation: Based on the Ct value of the sample, substitute it into the standard curve regression equation to calculate the copy number (copies / mL or copies / g) of gene recA in the sample to be tested, thereby obtaining the cell content of Hexanoic acid bacteria.

[0036] Preferably, the primer pair includes an upstream primer P4f (sequence shown in SEQ ID NO: 8) and a downstream primer P4r (sequence shown in SEQ ID NO: 9).

[0037] 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. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0038] Example 1: Selection of target strain and specific sequence.

[0039] In this embodiment of the invention, the bacterial strains to be tested are rumen bacteria (GDMCC 60133) or Lactobacillus lactis (GDMCC 1.1627). The morphology of rumen lactobacillus colonies is as follows: Figure 1 As shown, after culturing in RCM medium for 5–7 days, the colonies are milky white and round, with smooth edges and a raised center. Based on the genome sequences of *G. rumen caproic acid bacteria* (GDMCC 60133) and *G. rumen caproic acid bacteria* (GDMCC 1.1627) (both have identical recA gene sequences, both SEQ ID NO: 1), a search was conducted in the National Center for Biotechnology Information (NCBI) database to select commonly used housekeeping genes for taxonomy as candidate genes. The results are shown in Table 1.

[0040] Table 1 Comparison of housekeeping gene specificity

[0041]

[0042] The results showed that the recA gene sequence had the lowest similarity to other species (81.09%), exhibiting better specificity and being more suitable as a target sequence for qPCR detection than the commonly used 16S rRNA gene (96.83% similarity).

[0043] Example 2: Demonstration of detection effects of different primer pairs

[0044] 1. Primer design and specificity verification

[0045] Based on the recA sequence of the described hexanoic acid bacteria (as shown in SEQ ID NO: 1), 10 pairs of qPCR-specific primers were designed using Primer Premier 6, as shown in Table 2.

[0046] Table 2 qPCR primer sequences

[0047]

[0048] The specificity of the primers was verified using NCBI-Primer-BLAST; and the specificity of the primer pair was experimentally verified by PCR and agarose electrophoresis. The experimental subjects included: C6 bacterial DNA (Ruminocytosacchariformis, GDMCC 60133), C11 bacterial DNA (GenBank: KM454168.26, another rumen bacterium, used as a closely related reference control), mixed reference bacterial DNA (composed of equal viable amounts of Romboutsia lituseburensis, Bacillus cereus, Clostridium butyricum, Clostridium tyrobutyricum, Caproicibacterium amylolyticum, Ligilactobacillus acidipiscis, Clostridium kluyveri, and Clostridium sporogenes, used as a pure culture mixed reference control), and soil DNA (farmland soil, used as a reference control for uncultured microorganisms in complex environments).

[0049] qPCR reaction system (10 μL): 2 μL 2×SsoAdvanced Universal SYBR Green Supermix, 0.2 μL each of forward and reverse primers (10 μmol / L), 1 μL standard plasmid template, and 3.6 μL ddH2O. Reaction program: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 40 cycles.

[0050] The results are as follows Figure 2As shown. The results indicate that only the P4 primer pair (P4f and P4r) can specifically amplify the target hexanoic acid strain (…). Figure 2 The P4 primer pair (P4f and P4r) showed significant and stable band amplification (approximately 130–150 bp, consistent with the expected size), while no corresponding band amplification was observed for soil DNA, mixed reference bacteria, and closely related species C11. Other primer pairs showed amplification either for soil samples or for closely related species and mixed participating bacterial communities. This indicates that the P4 primer pair has significant species-level specificity and can be used for the specific identification and quantitative detection of target hexanoic acid bacteria (GDMCC 60133, GDMCC 1.1627).

[0051] Example 3: Quantitative Detection of Hexanoic Acid Bacteria

[0052] 1. Construction of recA gene recombinant plasmid

[0053] (1) Synthesis of short oligonucleotides: Based on the sequence information of the recA gene (SEQ ID NO: 1), 40-60 bp single-stranded short fragments were synthesized by the phosphoramidite method, and a 20-30 bp overlap region between adjacent fragments was designed.

[0054] (2) Assemble long target genes: short fragments are spliced ​​into genes of target length by overlapping extension PCR or ligase chain reaction.

[0055] (3) Enzyme digestion: The target gene (insertion fragment) and plasmid (vector) are digested with the same restriction endonuclease to obtain matching ends.

[0056] (4) Construction of recombinant plasmid: The digested insert fragment is ligated with T4 DNA to form a recombinant plasmid (WG94303_pUC57).

[0057] (5) Transformation of competent cells: The recombinant plasmid was introduced into competent bacteria by heat shock and then spread on LB plates containing the corresponding antibiotic (ampicillin) after recovery.

[0058] (6) Screening and Validation: Single colonies were picked from plates for amplification culture, plasmids were extracted, and preliminary validation was performed by enzyme digestion. The results are as follows: Figure 3 As shown, the results are consistent with expectations. Further sequencing confirmed that the recombinant plasmid (WG94303_pUC57) was correct, with a size of 3874 bp and a recA gene length of 1164 bp, which is 100% consistent with the expected gene sequence.

[0059] 2. Preparation of standard curve

[0060] (1) Plasmid concentration determination and copy number calculation: The concentration of the extracted recombinant recA plasmid was determined to be 130 ng / μL by UV spectrophotometer. The copy number was calculated according to the formula (total plasmid length is 3874 bp): Plasmid copy number = / (Total plasmid length (bp) × 660 g / mol / bp) = (copies / μL).

[0061] (2) Serial dilution: The standard plasmid was serially diluted 10-fold using ddH2O (10 7 10 6 10 5 10 4 10 3 (Copies / μL), with 3 replicates for each concentration, and temporary storage at 4°C (avoid repeated freeze-thaw cycles).

[0062] (3) qPCR amplification and standard curve plotting:

[0063] qPCR reaction system: 5 μL of 2×SsoAdvanced Universal SYBR Green Supermix, 0.2 μL each of the P4 forward and reverse primers (10 μmol / L) from Example 2, 1 μL of standard plasmid template (WG94303_pUC57), and 3.6 μL of ddH2O. Amplification program: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 40 cycles. After amplification, the temperature was increased from 60℃ to 95℃ at a rate of 0.5 °C / step, with a 2 s pause at each step and fluorescence was collected. The software plotted the fluorescence intensity against the first negative derivative of temperature (–dF / dT) to obtain the melting peak. The melting curve (as shown in the figure) was analyzed. Figure 4 As shown in the figure, a single sharp main peak was obtained (Tm is consistent with the theoretical value of recA gene (84.37℃)), indicating that the primer pair has good specificity.

[0064] A standard curve was generated using the qPCR instrument's built-in software, with the logarithm (lg) of the standard plasmid (WG94303_pUC57) copy number as the x-axis and Ct values ​​as the y-axis. The results are shown below. Figure 5 As shown. The equation of the standard curve is Ct = -3.17 × logt 10 (Initial template amount) +38.331, correlation coefficient (R²) is 0.998, amplification efficiency (E) is 106.5% (e.g. Figure 6 As shown in the figure, this indicates that the qPCR detection sensitivity is normal, the amplification efficiency is high, and the experimental results are reliable.

[0065] Example 3: Demonstration of the application effect of the detection method in the detection of fermentation system

[0066] Fermentation broth preparation: 1 mL of standard hexanoic acid bacteria (GDMCC 60133) solution was inoculated into 9 mL of RCM medium and anaerobic cultured at 37℃ for 24 h to obtain hexanoic acid bacteria seed culture. The hexanoic acid bacteria seed culture was inoculated into a bioreactor (RCM medium) at a 10% inoculation ratio and cultured for 2–3 days to obtain hexanoic acid bacteria liquid fermentation broth.

[0067] Genomic DNA extraction (using the Tiangen DP302 bacterial genomic DNA extraction kit in this example): Take 1-5 mL of *Hexanolysin* liquid fermentation broth, centrifuge at 10000 rpm for 1 min, and discard the supernatant. Resuspend in 200 μL of buffer GA, pretreat with lysozyme (50 mg / mL, 37℃, 30 min), add 20 μL of proteinase K, and mix well. Then add 220 μL of buffer GB, lyse at 70℃ for 10 min until the solution becomes clear. Add 220 μL of anhydrous ethanol, mix well, and transfer the entire solution to an adsorption column CB3. Centrifuge at 12000 rpm for 30 s and discard the filtrate. Then centrifuge sequentially with 500 μL of buffer GD and 600 μL of wash buffer PW (both with ethanol) for 30 s each, and discard the filtrate; finally, centrifuge an empty column for 1 min to remove residual liquid. Place CB3 in a new tube, add 50–200 μL of TE (pH 7–8.5) to the center of the membrane, incubate at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 2 minutes. The resulting solution is the genomic DNA and should be stored at -20°C for later use.

[0068] qPCR detection results: Following the method in Example 2, 1 μL of genomic DNA was used as a template, and qPCR was performed using specific primer pairs (P4 primer pairs P4f and P4r shown in Table 2). The number of cells in the sample was calculated using a standard curve as a reference. The results are shown in Table 3.

[0069] Table 3. qPCR detection results of Hexanoic acid bacteria in fermentation broth

[0070]

[0071] The results showed that the number of caproic acid bacteria in the two bioreactors was approximately 5.94 × 10⁻⁶. 10 ~5.96×10 10 The number of copies / mL indicates that Hexanoic acid bacteria have grown and proliferated extensively in the reactor.

[0072] Example 4: Demonstration of the application effect of the detection method in pit mud detection

[0073] All cellar mud samples used in this example were taken from the production workshop of a famous winery in Sichuan. Genomic DNA extraction from the cellar mud was performed using the Tiangen Soil Genomic DNA Extraction Kit (DP336): 0.5g of glass beads were added to a 15mL centrifuge tube. 0.4–0.5g of cellar mud sample was weighed and added to the same centrifuge tube. 1mL of Buffer SLX was added to the centrifuge tube, and the tube was vortexed at maximum speed for 3–5 minutes. 100μL of Buffer DS was added to the centrifuge tube, and the mixture was vortexed. The centrifuge tube was placed in a 70℃ water bath for 10 minutes. The centrifuge tube was then centrifuged at 3000rpm at room temperature for 3 minutes. 800μL of the supernatant was carefully transferred to a new 2mL centrifuge tube, and 270μL of Buffer SP2 was added, and the mixture was vortexed. The centrifuge tube was placed in an ice bath for 5 minutes, and then centrifuged at 4℃ and 13000xg for 5 minutes. Carefully transfer the supernatant to a new 2 mL centrifuge tube, add 0.7 volumes (approximately 600–900 μL) of isopropanol, and invert the tube 20–30 times to mix. Centrifuge at 13000 x g for 10 min at 4 °C to obtain DNA precipitate. Carefully remove the supernatant and invert the centrifuge tube on absorbent paper for 1 min. Add 200 μL of Elution Buffer and vortex for 10 s to dissolve the DNA. Add 100 μL of HTR Reagent and vortex for 10 s to mix. After standing at room temperature for 2 min, centrifuge at 13000 x g for 2 min at room temperature.

[0074] qPCR detection: Following the method in Example 2, 1 μL of template DNA was taken and detected by qPCR using specific primer pairs P4 (P4f and P4r). The colony count of the sample was calculated with reference to the standard curve. The results are shown in Table 4.

[0075] Table 4. qPCR detection results of caproic acid bacteria in pit mud.

[0076]

[0077] The results showed that the content of caproic acid bacteria in the 4-year-old fermentation pit was 3.15 × 10⁻⁶. 6 ~8.40×10 7 The content of caproic acid bacteria in a 50-year-old fermentation pit is approximately 1.21 × 10⁻⁶ copies / g, while the content of caproic acid bacteria in such pits is approximately 1.21 × 10⁻⁶ copies / g. 8 ~4.2×10 8 The results showed that the pit mud contained caproic acid bacteria (GDMCC60133, GDMCC 1.1627), and the content of caproic acid bacteria in the old pit was significantly higher than that in the new pit, which was consistent with the expectations.

[0078] Example 5: Demonstration of the application effect of the detection method in the detection of fermented mash

[0079] Genome extraction from fermented mash (using the Tiangen DP302 bacterial genomic DNA extraction kit in this example): All fermented mash samples in this example were taken from the production workshop of a famous winery in Sichuan. 1g of fermented mash was resuspended in 5mL of sterile water (vortexed for 1-2 min), then 3mL of the suspension was centrifuged at 10000rpm for 1 min, and the supernatant was discarded. 200μL of buffer GA was added for resuspending, and the mixture was pretreated with lysozyme (50mg / mL, 37℃, 30 min), followed by 20μL of proteinase K, and mixed well. Then, 220μL of buffer GB was added, and the mixture was lysed at 70℃ for 10 min until the solution became clear. 220μL of anhydrous ethanol was added and mixed well, then the entire mixture was transferred to an adsorption column CB3, centrifuged at 12000rpm for 30s, and the filtrate was discarded. The mixture was then centrifuged sequentially with 500μL buffer GD and 600μL wash buffer PW (both with added ethanol) for 30s each, and the filtrate was discarded; finally, the empty column was centrifuged for 1 min to remove residual liquid. Place CB3 in a new tube, add 50–200 μL of TE (pH 7–8.5) to the center of the membrane, incubate at room temperature for 2 minutes, then centrifuge at 12,000 rpm for 2 minutes. The resulting solution is the genomic DNA and should be stored at -20°C for later use.

[0080] qPCR results: 1 μL of template DNA was serially diluted 10–100 times with enzyme-free dd H2O water. Three samples were taken from each concentration. Following the method in Example 2, the samples were detected by qPCR with a standard curve as a reference, and the colony count was calculated. The results are shown in Table 5.

[0081] Table 5. qPCR detection results of Hexanoic acid bacteria in fermented mash.

[0082]

[0083] The content of caproic acid bacteria in the fermented mash was 2.20 × 10⁻⁶. 9 ~1.68×10 10 The number of copies / g indicates that the fermented mash contains the hexanoic acid bacteria (GDMCC 60133, GDMCC 1.1627) described in this patent.

[0084] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A pair of primers, characterized in that: The primer pair includes an upstream primer P4f, the sequence of which is shown in SEQ ID NO: 8, and a downstream primer P4r, the sequence of which is shown in SEQ ID NO:

9.

2. Detection reagents, test strips, or kits containing the primer pairs described in claim 1.

3. The use of the primer pair of claim 1 or the detection reagent, test strip or kit of claim 2 in the detection of caproic acid bacteria.

4. A method for detecting caproic acid bacteria, characterized in that: Based on the recA gene of the bacteria to be tested, a qPCR-specific primer pair was designed, and the bacteria were detected using the qPCR-specific primer pair.

5. The method according to claim 4, characterized in that: The primer pair includes an upstream primer P4f, the sequence of which is shown in SEQ ID NO: 8, and a downstream primer P4r, the sequence of which is shown in SEQ ID NO:

9.

6. The method according to claim 4 or 5, characterized in that: The method includes the following steps: (1) Design qPCR specific primer pairs based on the recA gene of the bacteria to be tested; (2) Extract total DNA from the sample to be tested and perform qPCR amplification detection using the primer pair; if a band appears, it is determined that the sample contains the hexanoic acid bacteria to be tested, and if no band appears, it is determined that the sample does not contain the hexanoic acid bacteria to be tested.

7. The method according to claim 6, characterized in that: The 10 μL qPCR amplification reaction system includes: 2 μL of 2×SsoAdvanced Universal SYBR Green Supermix, 0.2 μL each of forward and reverse primers, and 1 μL of standard plasmid template. 3.6μL.

8. The method according to claim 6, characterized in that: The qPCR amplification reaction program includes: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 30 s, 40 cycles.

9. The method according to claim 6, characterized in that: The method further includes steps (3) and (4): (3) Constructing the standard curve: (3-1) Plasmid concentration determination and copy number calculation: Determine the concentration of the standard plasmid and calculate the copy number; (3-2) Plotting the standard curve: Using the logarithm of the standard plasmid copy number as the abscissa and Ct value as the ordinate, generate the standard curve, calculate the regression equation and the correlation coefficient R²; (4) Quantitative detection of caproic acid bacteria: Substitute the Ct value of the sample to be tested into the regression equation of the standard curve to calculate the copy number of gene recA in the sample to be tested, thereby obtaining the cell content of caproic acid bacteria to be tested.

10. The method according to claim 8, characterized in that: The formula for calculating the copy number is: Copy number = The copy number is measured in copies / μL, and the total plasmid length is measured in bp.

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