Method for quantitatively detecting five kinds of lactobacillus PMA-qPCR viable bacteria based on new molecular target primers

Abstract

The invention discloses a method for quantitatively detecting five kinds of lactobacillus PMA-qPCR viable bacteria based on novel molecular target primers. The molecular target is as shown in SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3 and SEQ ID NO. 4. Compared with the prior art, the method has the advantages that viable count of single lactobacillus in a compound probiotic product can be achieved, the method has the advantages of being short in detection time, good in specificity, easy to operate and the like, a dye method and a probe method are established at the same time, and the method has a wider application scene and can better meet market requirements. Meanwhile, the counting method provided by the invention also overcomes the defects that the existing molecular detection method and plate counting method cannot distinguish the dead and live bacteria and cannot count the live but non-culturable bacteria, the counting result is more accurate, and the method is higher in practicability.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial detection, and particularly relates to a method for quantitatively detecting viable cells of five Lactobacillus species by PMA-qPCR based on new molecular target primers. Background Art

[0002] Lactobacillus is a group of Gram-positive, catalase-negative, spore-free, facultative anaerobic bacteria that can produce a large amount of lactic acid by fermenting carbohydrates. They are not only essential production strains for a variety of foods, but also exist in large numbers in the human digestive tract, and produce natural antibacterial substances such as organic acids, bacteriocins, and diacetyl through metabolism. They can play probiotic functions such as promoting intestinal health, improving cardiovascular function (assisting in reducing blood lipids), antioxidant activity, and antagonizing pathogenic bacteria by adjusting the intestinal flora and regulating immunity. Lactobacillus has played an important role in food fermentation since ancient times. With the discovery of its probiotic functions, it has been increasingly widely used in dairy products, infant foods, fermented meat products, dietary supplements, and functional foods. In recent years, Lactobacillus and its fermented foods have become a hot topic in food research. Since the efficacy of probiotic products mainly depends on the viability of the bacteria, the viable cell count of Lactobacillus in the products is the core content of the inspection of such foods. Therefore, a key factor for the continuous growth of the probiotic product market is the ability to accurately count the viable cells in any probiotic product, ensure that the viable cell count in the probiotic product can be maintained at a good level throughout the shelf life, and play a healthy role in the human body.

[0003] At present, the viable cell count results of probiotic products are mostly obtained by plate counting, that is, judging the life and death of bacteria based on their culturable properties. For example, the current "National Food Safety Standard for Microbiological Examination of Foods - Examination of Lactic Acid Bacteria GB 4789.35-2023" in China uses plate counting method and real-time fluorescence PCR method (qPCR) for identification. However, the plate counting method has disadvantages such as long time consumption (usually 72h), large workload, and cumbersome operation in practical applications. Although the qPCR method in the national standard gives full play to the high specificity of the qPCR method and can achieve qualitative detection of Lactobacillus, a quantitative method has not been established, and the advantages of the qPCR method have not been fully utilized.

[0004] In addition, there are also some rapid detection methods widely used in the detection of lactic acid bacteria in foods. Such as the ATP bioluminescence method, which can only count all bacteria with metabolic activity, and cannot detect a certain probiotic in a composite strain product. Similarly, there is flow cytometry. Although this method has high detection accuracy and can distinguish dead and live bacteria by the fluorescence permeability of dead and live bacteria, it cannot achieve counting of specific bacterial species, and this method has certain requirements for the test samples themselves and is also relatively expensive, with certain limitations.

[0005] Propidium monoazide bromide (PMA), as a reactive dye, can selectively penetrate the bacterial membrane of dead bacteria, covalently crosslink with its DNA, and hinder the amplification of its DNA, playing a role in distinguishing the living and dead of microorganisms. Excess reagent will react with water molecules under strong light irradiation and decompose into hydroxylamine compounds without crosslinking activity, making hydroxylamine no longer able to covalently crosslink DNA, thus avoiding affecting the subsequent DNA extraction and accurately achieving the counting of viable bacteria.

[0006] In recent years, a large number of patents or literatures at home and abroad have also used related technologies to detect, quantify or count viable bacteria of Lactobacillus helveticus, Limosilactobacillus fermentum, Lactiplantibacillus plantarum, Ligilactobacillus salivarius and Lactobacillus delbrueckii, which are specifically described as follows.

[0007] Although Patent CN 112029885 A screened specific molecular targets based on the pan-genome sequences of related strains, it only established a PCR method for its qualitative detection, but did not establish a quantitative or viable bacteria counting method. Patent CN 115820888 A is a live bacterial community analysis method established for biological samples, which has not been applied to the detection of probiotic products. At the same time, this method is more based on the analysis of bacterial communities and does not verify its accuracy. [2] GUO et al. [3] The PMA-qPCR method established by the people and related Patent CN117448467A has proven the accuracy of its counting through a large number of actual sample experiments, but this method can only count at the strain level and has a small scope of application. [4] Kim et al. [5] The people screened specific targets based on the 16s-23s rRNA sequence. The similarity between the 16s-23s rRNA sequences is relatively high, and the specificity is worse than the targets screened by the whole-genome analysis of the present invention, and viable bacteria counting cannot be performed. Ma Dan et al. [6] The research of the people designed primers based on the plnf gene. At the same time, this research only carried out artificial addition sample experiments and freeze-dried sample experiments of pure cultures, and did not test the products sold in the market. Shi et al. [7]Target screening was performed by humans based on the 16S rRNA gene and pheS gene of Lactobacillus delbrueckii subsp. bulgaricus, Lactiplantibacillus plantarum, Streptococcus thermophilus, Lactobacillus helveticus, and Lactococcus lactis subsp. lactis, and a standard curve was established by constructing plasmids. However, the accuracy of their counting was not verified in this study, and only the feasibility of the research method was demonstrated. Xu et al. [8] Humans also designed primers based on the 16S rRNA gene and pheS gene. This study mainly focused on the detection of rice flour products, and its scope of application was not wide. Duan Liangjie et al. [9] Humans and Herkenhoff et al.

[10] The targets and primers of the studies by humans were all from existing literature, and no new specific molecular targets were screened.

[0008] In summary, the existing viable cell counting methods for probiotics still have many deficiencies in terms of target specificity, detection specificity, discrimination between live and dead bacteria, counting accuracy, and scope of method application. Therefore, exploring new molecular targets and establishing a viable cell counting method with a wide scope of application and high counting accuracy have become important aspects of this invention. Summary of the Invention

[0009] Aiming at the deficiencies of the existing Lactobacillus quantification methods, the purpose of this invention is to provide a method that can quickly and accurately perform viable cell quantification detection on Lactobacillus helveticus, Lactobacillus mucosae, Lactiplantibacillus plantarum, Ligilactobacillus salivarius, and Lactobacillus delbrueckii.

[0010] Therefore, based on pan-genome analysis, this invention has explored new molecular targets and combined the qPCR method with PMA dye to establish the PMA-qPCR method. This invention can achieve highly specific viable cell counting, solve problems such as inaccurate counting results, inability to count single strains in composite probiotic products, inability to distinguish between live and dead bacteria, and narrow scope of method application. Moreover, this invention has established both the dye method and the probe method at the same time, making the application scenario of this invention broader and better meeting the market demand.

[0011] The purpose of this invention is to provide a specific molecular target for identifying Lactobacillus helveticus, Lactobacillus mucosae, Lactiplantibacillus plantarum, Ligilactobacillus salivarius, and Lactobacillus delbrueckii.

[0012] Based on whole-genome comparative analysis, this invention has screened specific molecular targets for Lactobacillus mucosae, Lactiplantibacillus plantarum, Ligilactobacillus salivarius, and Lactobacillus delbrueckii, and the target information is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.

[0013] The second object of the present invention is to provide primer pairs and probes that can specifically detect Lactobacillus helveticus and the above-mentioned specific molecular targets through the design and screening of specific primers. The nucleotide sequences of the primers and probes used for detection are shown in SEQ ID NOs. 5-21.

[0014] SEQ ID NO.5 and SEQ ID NO.6 are primer pairs used for detection of Lactobacillus helveticus by dye method; SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 are primer pairs and probes used for detection of Lactobacillus helveticus by probe method; SEQ ID NO.8 and SEQ ID NO.9 are primer pairs used for detection of fermented mucus lactobacillus by dye method; SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 are primer pairs and probes used for detection of fermented mucus lactobacillus by probe method; SEQ ID NO.11 and SEQ ID NO.12 are primer pairs used for detection of plant lactobacillus by dye method; SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13 are primer pairs and probes used for detection of plant lactobacillus by probe method; SEQ ID NO.14 and SEQ ID NO.15 are primer pairs used for detection of saliva combined lactobacillus by dye method; SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16 is a primer pair and a probe used for the saliva combined Lactobacillus probe method detection; SEQ ID NO.17 and SEQ ID NO.18 are primer pairs used for the Lactobacillus delbrueckii dye method detection; SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21 are primer pairs and a probe used for the Lactobacillus delbrueckii probe method detection.

[0015] The third object of the present invention is to provide a PMA-qPCR live bacteria quantitative detection method for five types of lactobacilli, which uses the above-mentioned primers and / or probes to detect the microorganisms to be detected.

[0016] Preferably, the method includes a dye method and a probe method, and the steps are as follows:

[0017] a. Treat the sample to be tested with 30-50 μmol / L PMA to obtain a PMA-treated sample to be tested;

[0018] b. Use a DNA extraction kit to extract the genomic DNA of the sample to be tested, and use the above primers to perform qPCR amplification of the DNA of the sample to be tested by the dye method or probe method;

[0019] c The logarithmic value of the concentration of the pure culture of the gradient diluted bacterial solution is used as the horizontal axis, and the corresponding qPCR fluorescence signal value is used as the vertical axis. The fitted curve is the standard curve used for counting comparison;

[0020] Substitute the fluorescence signal value generated by amplifying the sample into the standard curve equation for calculation to determine the viable count of Lactobacillus in the sample.

[0021] Preferably, the dye-based qPCR amplification system is as follows: 10 μL of TB Green Premix reaction solution, 2 μL of template DNA, 0.4 - 0.8 μL each of forward and reverse primers at 10 μmol / L, and ddH2O is added to make up the total volume to 20 μL; the dye-based qPCR program is as follows: in the first stage, pre-denaturation at 95°C for 30 - 300 S; in the second stage, denaturation at 95°C for 5 S, annealing at 60 - 65°C for 20 - 30 S and collect fluorescence signals or denaturation at 95°C for 5 S, annealing at 60 - 65°C for 20 - 30 S, extension at 72°C for 20 - 30 S, for a total of 40 cycles; in the third stage, 60 S at 95°C, 60 S at 40°C, 1 S at 65°C, 1 S at 97°C for a total of one cycle;

[0022] The probe-based qPCR amplification system is as follows: 10 μL of Premix Ex Taq reaction solution, 2 μL of template DNA, 0.4 - 0.8 μL each of forward and reverse primers and probe at 10 μmol / L, and ddH2O is added to make up the total volume to 20 μL; the probe-based qPCR program is as follows: in the first stage, pre-denaturation at 95°C for 30 S; in the second stage, denaturation at 95°C for 5 S, annealing at 60 - 65°C for 20 - 30 S and collect fluorescence signals, for a total of 40 cycles.

[0023] The fifth object of the present invention is to provide a kit for quantitatively determining viable bacteria of Lactobacillus helveticus, Lactobacillus mucosae, Lactobacillus plantarum, Lactobacillus salivarius and Lactobacillus delbrueckii. The kit includes DNA extraction reagents, primer pairs and / or probes used in the dye-based method for the corresponding strains, PMA dye, qPCR premix, negative control products and positive control products.

[0024] This kit can be used for viable bacteria counting of Lactobacillus helveticus, Lactobacillus mucosae, Lactobacillus plantarum, Lactobacillus salivarius and Lactobacillus delbrueckii in lactic acid bacteria culture solution, probiotic solid beverage, lactic acid bacteria freeze-dried powder and lactic acid bacteria fermented milk.

[0025] Advantages of the present invention

[0026] The present invention provides a PMA-qPCR method for detecting and achieving viable cell counting of Lactobacillus helveticus, Lactobacillus fermentum, Lactiplantibacillus plantarum, Ligilactobacillus salivarius, and Lactobacillus delbrueckii, as well as the molecular targets, primer pairs, and probes used thereby. Compared with the existing technologies, the present invention can achieve viable cell counting of a single lactobacillus in a compound probiotic product, and has the characteristics of short detection time, good specificity, and simple operation. Meanwhile, the present invention has established both a dye method and a probe method, with a broader application scenario, and can better meet the market demand. At the same time, the counting method of the present invention also makes up for the deficiencies of the existing molecular detection methods and plate counting methods in being unable to distinguish between live and dead bacteria and being unable to count viable but non-culturable bacteria, with more accurate counting results and stronger practicality of the method. Description of the Drawings

[0027] Figure 1 are the specific detection results and quantitative standard curves of the PMA-qPCR dye method for Lactobacillus helveticus;

[0028] Figure 2 are the specific detection results and quantitative standard curves of the PMA-qPCR probe method for Lactobacillus helveticus;

[0029] Figure 3 are the specific detection results and quantitative standard curves of the PMA-qPCR dye method for Lactobacillus fermentum;

[0030] Figure 4 are the specific detection results and quantitative standard curves of the PMA-qPCR probe method for Lactobacillus fermentum;

[0031] Figure 5 are the specific detection results and quantitative standard curves of the PMA-qPCR dye method for Lactiplantibacillus plantarum;

[0032] Figure 6 are the specific detection results and quantitative standard curves of the PMA-qPCR probe method for Lactiplantibacillus plantarum;

[0033] Figure 7 are the specific detection results and quantitative standard curves of the PMA-qPCR dye method for Ligilactobacillus salivarius;

[0034] Figure 8 are the specific detection results and quantitative standard curves of the PMA-qPCR probe method for Ligilactobacillus salivarius;

[0035] Figure 9 are the specific detection results and quantitative standard curves of the PMA-qPCR dye method for Lactobacillus delbrueckii;

[0036] Figure 10 are the specific detection results and quantitative standard curves of the PMA-qPCR probe method for Lactobacillus delbrueckii;

[0037] Figure 11 are the results of plate counting, qPCR amplification curve, and MALDI-TOF identification of the Lactobacillus helveticus kit by Experimenter A;

[0038] Figure 12 are the results of plate counting, qPCR amplification curve, and MALDI-TOF identification of the Lactobacillus mucosae fermentans kit by Experimenter A;

[0039] Figure 13 are the results of plate counting, qPCR amplification curve, and MALDI-TOF identification of the Lactiplantibacillus plantarum kit by Experimenter A;

[0040] Figure 14 are the results of plate counting, qPCR amplification curve, and MALDI-TOF identification of the Ligilactobacillus salivarius kit by Experimenter A;

[0041] Figure 15 are the results of plate counting, qPCR amplification curve, and MALDI-TOF identification of the Lactobacillus delbrueckii kit by Experimenter A;

[0042] Figure 16 are the results of plate counting and qPCR amplification curve of the Lactobacillus helveticus kit by Experimenter B;

[0043] Figure 17 are the results of plate counting and qPCR amplification curve of the Lactobacillus mucosae fermentans kit by Experimenter B;

[0044] Figure 18 are the results of plate counting and qPCR amplification curve of the Lactiplantibacillus plantarum kit by Experimenter B;

[0045] Figure 19 are the results of plate counting and qPCR amplification curve of the Ligilactobacillus salivarius kit by Experimenter B;

[0046] Figure 20 are the results of plate counting and qPCR amplification curve of the Lactobacillus delbrueckii kit by Experimenter B. Detailed implementation manners

[0047] To more clearly illustrate the purpose, technical features, and beneficial effects of the present invention, the present invention will be further described in conjunction with the accompanying drawings and specific embodiments.

[0048] Example 1 Screening of Lactobacillus-specific molecular targets and design of specific primers

[0049] Download the genomic sequences of bacteria belonging to the genus Lactobacillus but different species on the NCBI website (http: / / www.ncbi.nlm.nih.gov / ), and then screen for specific molecular detection targets of common target strains through pan-genome analysis methods. Specific method: Annotate all genomic sequences used for analysis through the PGAP software, then copy the annotated gff files of all strains and perform pan-genome analysis. After running, the generated gene_presence_absence.csv file is the matrix file of the pan-genome. Then select sequences that are 100% present in the genomic sequences of all target strains but absent in the genomes of other non-target strains as targets. Then perform Blast alignment of the obtained target genes on NCBI, and finally select fragments with good specificity as detection targets. The nucleotide sequences of the specific molecular targets of Lactobacillus fermentum, Lactiplantibacillus plantarum, Ligilactobacillus salivarius, and Lactobacillus delbrueckii finally screened by the present invention are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4.

[0050] According to the nucleotide sequence information of the above targets and the existing patent targets, use Primer Premier 5.0 to design specific primers and probes, and then perform primer Blast on the NCBI website for the designed primers and probes to select primer pairs with better specificity. Finally, the primer and probe information screened by the present invention is shown as SEQ ID NO.5-21.

[0051] Among them, SEQ ID NO.5 and SEQ ID NO.6 are the primer pairs for the dye method detection of Lactobacillus helveticus; SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 are the primer pairs and probe for the probe method detection of Lactobacillus helveticus; SEQ ID NO.8 and SEQ ID NO.9 are the primer pairs for the dye method detection of Lactobacillus mucosae; SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 are the primer pairs and probe for the probe method detection of Lactobacillus mucosae; SEQ ID NO.11 and SEQ ID NO.12 are the primer pairs for the dye method detection of Lactiplantibacillus plantarum; SEQ ID NO.11, SEQ ID NO.12 and SEQ ID NO.13 are the primer pairs and probe for the probe method detection of Lactiplantibacillus plantarum; SEQ ID NO.14 and SEQ ID NO.15 are the primer pairs for the dye method detection of Ligilactobacillus salivarius; SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO.16 are the primer pairs and probe for the probe method detection of Ligilactobacillus salivarius; SEQ ID NO.17 and SEQ ID NO.18 are the primer pairs for the dye method detection of Lactobacillus delbrueckii; SEQ ID NO.19, SEQ ID NO.20 and SEQ ID NO.21 are the primer pairs and probe for the probe method detection of Lactobacillus delbrueckii.

[0052] The probes SEQ ID NO.7, SEQ ID NO.10, SEQ ID NO.13, SEQ ID NO.16, SEQ ID NO.21 of the present invention are all Taqman probes, with a FAM fluorescent reporter group connected to the 5' end of the probe and a BHQ1 fluorescent quenching group connected to the 3' end. The fluorescent reporter group of the present invention can be a fluorescent reporter group commonly used in the art, such as FAM, HEX or VIC, etc.; similarly, the fluorescent quenching group can also be a fluorescent quenching group commonly used in the art, such as BHQ3, BHQ1, BHQ2 or TAMRA, etc.

[0053] Example 2 Specificity evaluation of primers and probes for five Lactobacillus strains

[0054] Take 17 Lactobacillus strains (strain information is shown in Table 3), including 1 strain of Lactobacillus helveticus, Lactobacillus mucosae, Lactiplantibacillus plantarum, Ligilactobacillus salivarius and Lactobacillus delbrueckii respectively, and 12 other Lactobacillus strains, and conduct specificity verification on the screened primers and probes. The specific steps are as follows:

[0055] Take 500 μL of bacterial culture in a 1.5 mL centrifuge tube, centrifuge at 12000 r / min for 1 min, discard the supernatant, collect the bacteria, and extract the DNA therein as template DNA according to the extraction operation instructions of Gram-positive bacteria in the DNA extraction kit of Guangdong Huankai Biotechnology Co., Ltd.

[0056] With Roche Light The samples were amplified on a 96 fluorescence quantitative PCR instrument. The primers and probes used were consistent with those in Example 1 and were synthesized by Beijing Liuhe BGI Gene Technology Co., Ltd.

[0057] The qPCR dye method amplification system and procedure are shown in Table 1 below, and the qPCR probe method amplification system and procedure are shown in Table 2 below.

[0058] Table 1 Dye-based amplification system and procedures

[0059]

[0060] Table 2 Probe method amplification system and procedures

[0061]

[0062] Exploiting software 96SW 1.1 Check the experimental results. When the blank control does not produce a fluorescent signal, if the Cq of the sample is greater than 35 cycles, it means that the sample does not contain the target bacteria; if the Cq value is less than 35 cycles, it means that the sample contains the target bacteria. The number of live bacteria of the target bacteria in the system can be quantitatively detected by the fluorescent signal value in the system.

[0063] The results showed that the screened probes and primer sets had obvious S-shaped amplification curves for positive target strains, and had no or no obvious S-shaped amplification curves for other negative non-target strains, that is, all primer sets and probes had good specificity.

[0064] The target bacteria and non-target bacteria used were identified by MALDI-TOF mass spectrometry, and their types and numbers are shown in Table 3. Figure 1 As shown above, the results of the probe specificity experiment are as follows Figure 2 As shown above; the specific experimental results of the fermented mucus lactobacillus dye method are as follows Figure 3 As shown above, the results of the probe specificity experiment are as follows Figure 4 As shown above; the specific experimental results of the Lactobacillus plantarum dye method are as follows Figure 5 As shown above, the results of the probe specificity experiment are as follows Figure 6 As shown above; the specific experimental results of saliva combined with Lactobacillus dye method are as follows Figure 7As shown above, the results of the specificity experiment by the probe method are as Figure 8 shown above; the results of the specificity experiment by the dye method for Lactobacillus delbrueckii are as Figure 9 shown above, and the results of the specificity experiment by the probe method are as Figure 10 shown above. The curves in the figure show that the target bacteria were detected, and there was no amplification curve for the remaining non-target bacteria, which is the flat line below.

[0065] Table 3 Information on the strains used in the primer specificity experiment

[0066]

[0067]

[0068] Example 3 Preparation of standard products and construction of standard curves for the quantitative detection method of five Lactobacillus live bacteria

[0069] The five Lactobacillus strains cultured overnight were respectively diluted in ten-fold gradients using PBS buffer to obtain pure culture bacterial solutions in ten-fold gradients, and their plate counts were performed with reference to the method of "GB 4789.35-2023 National Food Safety Standard Food Microbiology Examination - Lactic Acid Bacteria Examination".

[0070] 7.5, 10, 10, 12.5, and 10 μL of PMA solution with a concentration of 2 mmol / L were respectively added to the ten-fold gradient pure culture bacterial solution samples of Lactiplantibacillus plantarum, Lactobacillus mucosae fermentans, Lactobacillus helveticus, Ligilactobacillus salivarius, and Lactobacillus delbrueckii. The centrifuge tubes were placed in an incubation box and incubated for 15 min, and during the incubation, the incubation box was kept on a sample mixer. After the dark incubation ended, the centrifuge tubes were placed under a 650W tungsten filament lamp for light irradiation for 15 min and placed on ice during the light irradiation. The treated samples were centrifuged at 12000 r / min for 1 min, the supernatant was discarded, and they were washed once with 1 mL of PBS buffer and the precipitate was retained after centrifuging at 12000 r / min for 1 min.

[0071] The method for preparing the DNA template was the same as that in Example 2, and the DNA extracted was the standard product for this method. The methods for qPCR amplification and reading qPCR results were both the same as those in Example 2.

[0072] Taking the value after taking the Log 10 value of the plate count result as the abscissa and the fluorescence signal value of the standard product as the ordinate to establish a standard curve, and the regression coefficients R 2 of the standard curves of the five Lactobacillus PMA-qPCR (dye method and probe method) methods established were all greater than 0.99.

[0073] Among them, the standard curve of the dye method for Lactobacillus helveticus is as Figure 1 shown below, and the standard curve of the probe method is as Figure 2as shown below; the standard curve of Lactobacillus mucosae by the dye method is as Figure 3 shown below, and the standard curve of the probe method is as Figure 4 shown below; the standard curve of Lactiplantibacillus plantarum by the dye method is as Figure 5 shown below, and the standard curve of the probe method is as Figure 6 shown below; the standard curve of Ligilactobacillus salivarius by the dye method is as Figure 7 shown below, and the standard curve of the probe method is as Figure 8 shown below; the standard curve of Lactobacillus delbrueckii by the dye method is as Figure 9 shown below, and the standard curve of the probe method is as Figure 10 shown below.

[0074] Detection of actual samples in Example 4

[0075] Weigh 1 g (mL) of the probiotic product sold on the market, add it to 9 mL of PBS buffer, vortex thoroughly until there is no obvious precipitate, and dilute the total sample concentration to about 10 7 CFU / mL. Take 500 μL of the diluted sample for subsequent experiments. If the sample to be tested can be plate counted, refer to the method of "GB 4789.35-2023 National Food Safety Standard Microbiological Examination of Foods - Examination of Lactic Acid Bacteria" to perform plate counting on it.

[0076] The PMA treatment method is the same as that in Example 3, and the methods of DNA template preparation, qPCR amplification, and qPCR result reading are the same as those in Example 2. When performing qPCR amplification, set a positive control group and a negative control group. The DNA template of the positive control group is the DNA extracted from the pure culture broth of the target bacteria, and the DNA template of the negative control group is sterile water.

[0077] Substitute the fluorescence signal value of the sample to be tested into the standard curve to calculate its viable count. The results are shown in Table 4. For the established PMA-qPCR method, whether it is the dye method or the probe method, the difference from the plate count or the labeled bacterial content of the product is small, that is, the counting result of this method has good accuracy.

[0078] Table 4 Detection results of actual samples

[0079]

[0080]

[0081] Kit proficiency testing in Example 5

[0082] Different experimenters (A, B) used the kit described in the present invention to test the same probiotic product sold on the market.

[0083] Weigh 1 g (mL) of each probiotic product sold on the market, add it to 9 mL of PBS buffer, vortex thoroughly until there is no obvious precipitation, and dilute the total sample concentration to approximately 10 7 CFU / mL. Take 500 μL of the diluted sample for subsequent experiments and perform plate counting simultaneously. Among them, the plate counting results of the Lactobacillus helveticus sample by Experimenter A are as shown in Figure 11 Figure a, the plate counting results of the Lactobacillus mucosae sample are as shown in Figure 12 Figure a, the plate counting results of the Lactiplantibacillus plantarum sample are as shown in Figure 13 Figure a, the plate counting results of the Ligilactobacillus salivarius sample are as shown in Figure 14 Figure a, the plate counting results of the Lactobacillus delbrueckii sample are as shown in Figure 15 Figure a. The plate counting results of the Lactobacillus helveticus sample by Experimenter B are as shown in Figure 16 Figure a, the plate counting results of the Lactobacillus mucosae sample are as shown in Figure 17 Figure a, the plate counting results of the Lactiplantibacillus plantarum sample are as shown in Figure 18 Figure a, the plate counting results of the Ligilactobacillus salivarius sample are as shown in Figure 19 Figure a, the plate counting results of the Lactobacillus delbrueckii sample are as shown in Figure 20 Figure a.

[0084] After culturing for 48 h, use MALDI-TOF mass spectrometry to identify the colonies on the plate to determine the concentration of various bacteria in the sample. Since the results of different experimenters using MALDI-TOF mass spectrometry are the same, they are not shown separately. Among them, the MALDI-TOF identification results of the Lactobacillus helveticus sample are as shown in Figure 11 Figure b, the colony morphology is white and saturated, which is Lactobacillus helveticus; the MALDI-TOF identification results of the Lactobacillus mucosae sample are as shown in Figure 12 Figure b, the colonies are small and the surface is white, which is Lactobacillus mucosae; the MALDI-TOF identification results of the Lactiplantibacillus plantarum sample are as shown in Figure 13 Figure b, the colonies are smooth and round, which is Lactiplantibacillus plantarum; the MALDI-TOF identification results of the Ligilactobacillus salivarius sample are as shown in Figure 14 Figure b, the colony morphology is white and saturated, which is Ligilactobacillus salivarius; the MALDI-TOF identification results of the Lactobacillus delbrueckii sample are as shown in Figure 15 Figure b, the colonies are large, plump and round, which is Lactobacillus delbrueckii.

[0085] Add 7.5, 10, 10, 12.5, and 10 μL of PMA with a concentration of 2 mmol / L to the samples of Lactiplantibacillus plantarum, Limosilactobacillus fermentum, Lactobacillus helveticus, Ligilactobacillus salivarius, and Lactobacillus delbrueckii, respectively. Place the centrifuge tubes in an incubation box and incubate for 15 min. During the incubation, keep the incubation box on a sample mixer. After the dark incubation, place the centrifuge tubes under a 650 W tungsten filament lamp for 15 min, and place them on ice during the illumination. Centrifuge the treated samples at 12,000 r / min for 1 min, discard the supernatant, add 1 mL of PBS buffer to wash once, and centrifuge at 12,000 r / min for 1 min to retain the precipitate. Then, prepare the template DNA according to the extraction operation instructions in the DNA extraction kit of Guangdong Huankai Microbial Science & Technology Co., Ltd.

[0086] Use Roche Perform qPCR dye method and probe method amplifications on the template DNA using a Roche 96 fluorescence quantitative PCR instrument. When performing the amplification, set up positive and negative control groups respectively. The DNA template of the positive control group is reagent B, and the DNA template of the negative control group is sterile water. The amplification procedures and reaction systems of the dye method and probe method are the same as those in Example 2. Among them, the qPCR dye method amplification curve results of the Lactobacillus helveticus sample of Experimenter A are as shown in Figure 11 c, and the probe method amplification curve results are as shown in Figure 11 d; the qPCR dye method amplification curve results of the Limosilactobacillus fermentum sample are as shown in Figure 12 c, and the probe method amplification curve results are as shown in Figure 12 d; the qPCR dye method amplification curve results of the Lactiplantibacillus plantarum sample are as shown in Figure 13 c, and the probe method amplification curve results are as shown in Figure 13 d; the qPCR dye method amplification curve results of the Ligilactobacillus salivarius sample are as shown in Figure 14 c, and the probe method amplification curve results are as shown in Figure 14 d; the qPCR dye method amplification curve results of the Lactobacillus delbrueckii sample are as shown in Figure 15 c, and the probe method amplification curve results are as shown in Figure 15 d. The qPCR dye method amplification curve results of the Lactobacillus helveticus sample of Experimenter B are as shown in Figure 16 b, and the probe method amplification curve results are as shown in Figure 16 c; the qPCR dye method amplification curve results of the Limosilactobacillus fermentum sample are as shown in Figure 17 b, and the probe method amplification curve results are as shown in Figure 17 c; the qPCR dye method amplification curve results of the Lactiplantibacillus plantarum sample are as shown in Figure 18 b, and the probe method amplification curve results are as shown in Figure 18 c; the qPCR dye method amplification curve results of the Ligilactobacillus salivarius sample are as shown in Figure 19As shown in Figure b, the amplification curve results of the probe method are as follows Figure 19 As shown in Figure c; the amplification curve results of the qPCR dye method for Lactobacillus delbrueckii samples are as follows Figure 20 As shown in Figure b, the amplification curve results of the probe method are as follows Figure 20 As shown in Figure c.

[0087] The fluorescence signal values obtained by qPCR were substituted into the standard curve to calculate the viable cell count, and the results are shown in Table 5. The results show that for the established PMA-qPCR method, whether it is the dye method or the probe method, the difference from the plate count or the bacteria content marked on the product is small, that is, the counting results of this method have good accuracy.

[0088] Table 5 Results of the proficiency testing experiment of the kit

[0089]

[0090] SEQ ID NO.1:

[0091]

[0092]

[0093] SEQ ID NO.2:

[0094] SEQ ID NO.3:

[0095] SEQ ID NO.4:

[0096]

[0097] SEQ ID NO.5:

[0098] tgccacaaag gttccgaat 19

[0099] SEQ ID NO.6:

[0100] gcatctaatg gaagaagcgt ga 22

[0101] SEQ ID NO.7:

[0102] atggatcagt cgtgctacta tcaac 25

[0103] SEQ ID NO.8:

[0104] tactttcacc gccgctacct 20

[0105] SEQ ID NO.9:

[0106] cgtgcttggt cactgattgg t 21

[0107] SEQ ID NO.10:

[0108] ttgcaggagc taaaccacaa tctatc 26

[0109] SEQ ID NO.11:

[0110] gttaattggt ggggcgtttg 20

[0111] SEQ ID NO.12:

[0112] tgctgataag ggtgctcata gg 22

[0113] SEQ ID NO.13:

[0114] ctcgccagac gttatatcgc tt 22

[0115] SEQ ID NO.14:

[0116] tcccaccaga tgttgaggat 20

[0117] SEQ ID NO.15:

[0118] acatcaccgc cagatacagg 20

[0119] SEQ ID NO.16:

[0120] ttgcaggagc taaaccacaa tctatc 26

[0121] SEQ ID NO.17:

[0122] gcaagtgaaa gaagcggttg 20

[0123] SEQ ID NO.18:

[0124] tggtagctgg gtccatagag 20

[0125] SEQ ID NO.19:

[0126] ggcaggatta gccgaaggaa a 21

[0127] SEQ ID NO.20:

[0128] cagggtcttg gtcgtgactc t 21

[0129] SEQ ID NO.21:

[0130] gctggacaag taccggctgg gaccg 25

Claims

1. A specific molecular target for identifying Lactobacillus helveticus, Lactobacillus mucosae, Lactiplantibacillus plantarum, Ligilactobacillus salivarius, and Lactobacillus delbrueckii, characterized in that, The molecular targets are as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.

4.

2. A primer pair and a probe for identifying Lactobacillus helveticus, Lactobacillus mucosae, Lactobacillus plantarum, Lactobacillus salivarius subsp. salicinius, and Lactobacillus delbrueckii, characterized in that, The nucleotide sequences of the primer pairs and probes used for detection are as shown in SEQ ID NO.5 to 21. Among them, SEQ ID NO.5 and SEQ ID NO.6 are the primer pair used for the detection of Lactobacillus helveticus by the dye method; SEQ ID NO.5, SEQ ID NO.6, and SEQ ID NO.7 are the primer pair and probe used for the detection of Lactobacillus helveticus by the probe method; SEQ ID NO.8 and SEQ ID NO.9 are the primer pair used for the detection of Lactobacillus mucosae by the dye method; SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10 are the primer pair and probe used for the detection of Lactobacillus mucosae by the probe method; SEQ ID NO.11 and SEQ ID NO.12 are the primer pair used for the detection of Lactobacillus plantarum by the dye method; SEQ ID NO.11, SEQ ID NO.12, and SEQ ID NO.13 are the primer pair and probe used for the detection of Lactobacillus plantarum by the probe method; SEQ ID NO.14 and SEQ ID NO.15 are the primer pair used for the detection of Lactobacillus salivarius by the dye method; SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16 are the primer pair and probe used for the detection of Lactobacillus salivarius by the probe method; SEQ ID NO.17 and SEQ ID NO.18 are the primer pair used for the detection of Lactobacillus delbrueckii by the dye method; SEQ ID NO.19, SEQ ID NO.20, and SEQ ID NO.21 are the primer pair and probe used for the detection of Lactobacillus delbrueckii by the probe method.

3. A PMA-qPCR viable cell quantification method for Lactobacillus helveticus, Lactobacillus mucosae, Lactiplantibacillus plantarum, Ligilactobacillus salivarius and Lactobacillus delbrueckii, characterized in that, It is to detect the microorganism to be tested with the primer pair and / or probe described in claim 2.

4. The method according to claim 3, wherein It includes the dye method and the probe method, and the steps are as follows: a. Treat the sample to be tested with 30 - 50 μmol / L of PMA to obtain the sample to be tested after PMA treatment. b. Extract the genomic DNA of the sample to be tested using a DNA extraction kit, and perform qPCR amplification of the DNA of the sample to be tested by the dye method or the probe method using the above primers. c. Taking the logarithm of the concentration of the pure culture of the bacterial liquid diluted in gradient as the abscissa and the corresponding fluorescence signal value of qPCR as the ordinate, the fitted curve is the standard curve for counting comparison. d. Substitute the fluorescence signal value generated by the amplification of the sample into the standard curve equation for calculation to determine the viable count of Lactobacillus in the sample.

5. The method according to claim 4, wherein The qPCR amplification system of the dye method is: 10 μL of TBGreen Premix reaction solution, 2 μL of template DNA, 0.4 - 0.8 μL of each of the forward and reverse primers at 10 μmol / L, and ddH2O is added to make up the total volume to 20 μL; the qPCR program of the dye method is: in the first stage, pre-denaturation at 95°C for 30 - 300 S; The second stage: denaturation at 95°C for 5 s, annealing at 60 - 65°C for 20 - 30 s and collecting fluorescence signals, or denaturation at 95°C for 5 s, annealing at 60 - 65°C for 20 - 30 s, extension at 72°C for 20 - 30 s, for a total of 40 cycles; The third stage: one cycle of 60 s at 95°C, 60 s at 40°C, 1 s at 65°C, and 1 s at 97°C; 6. The method according to claim 4, wherein The probe-based qPCR amplification system is as follows: 10 μL of Premix ExTaq reaction solution, 2 μL of template DNA, 0.4 - 0.8 μL each of forward and reverse primers and probe at 10 μmol / L, and ddH2O is added to make up the total volume to 20 μL; The probe-based qPCR program is as follows: The first stage: pre-denaturation at 95°C for 30 s; The second stage: denaturation at 95°C for 5 s, annealing at 60 - 65°C for 20 - 30 s and collecting fluorescence signals, for a total of 40 cycles.

7. A kit for quantitatively determining viable bacteria of Lactobacillus helveticus, Lactobacillus mucosae, Lactobacillus plantarum, Lactobacillus salivarius, and Lactobacillus delbrueckii, characterized in that, The kit includes DNA extraction reagents, primer pairs and / or probes used in the dye method for the corresponding bacterial species, PMA dye, qPCR premix, negative control product and positive control product.

Images