Fluorescent quantitative PCR (Polymerase Chain Reaction) primer and application thereof in counting of probiotics in mixed fermentation

Abstract

The invention provides a fluorescent quantitative PCR (Polymerase Chain Reaction) primer and application thereof in counting of probiotics in mixed fermentation. The fluorescent quantitative PCR primer comprises a fluorescent quantitative PCR primer of staphylococcus xylosus, a fluorescent quantitative PCR primer of lactobacillus fermentum and a fluorescent quantitative PCR primer of phytobacterium plantarum, wherein the fluorescent quantitative PCR primer comprises a fluorescent quantitative PCR primer of staphylococcus xylosus, a fluorescent quantitative PCR primer of lactobacillus fermentum and a fluorescent quantitative PCR primer of lactobacillus plantarum; an upstream primer of the fluorescent quantitative PCR primer of the staphylococcus xylosus is as shown in SEQ ID No. 1, and a downstream primer of the fluorescent quantitative PCR primer of the staphylococcus xylosus is as shown in SEQ ID No. 2; an upstream primer of the fluorescent quantitative PCR primer of the lactobacillus fermentum is as shown in SEQ ID No.3, and a downstream primer of the fluorescent quantitative PCR primer of the lactobacillus fermentum is as shown in SEQ ID No.4; an upstream primer of the fluorescent quantitative PCR primer of the phytobacterium plantarum is shown as SEQ ID No.5, and a downstream primer of the fluorescent quantitative PCR primer of the phytobacterium plantarum is shown as SEQ ID No.6. According to the primer group, primer amplification fragments are shortened, so that the result obtained in a fluorescent quantitative PCR experiment is more accurate, the influence of template integrity change on a target fragment is reduced, the appearance time can be shortened, and the Cq value is reduced.

Description

Technical Field

[0001] The invention relates to a fluorescent quantitative PCR primer and application thereof in probiotics counting in mixed bacteria fermentation, belonging to the technical field of microorganisms. Background Art

[0002] People's use of microorganisms has transitioned from natural mixed bacterial culture to pure bacterial culture, reducing the complex problems of coexistence of multiple microorganisms. However, in subsequent research and experiments, it was found that many important metabolic and biochemical processes cannot be completed or can only be carried out weakly by a single strain, and must rely on the co-cultivation of two or more microorganisms to complete the metabolic and biochemical process, that is, mixed bacterial fermentation.

[0003] In recent years, mixed bacteria fermentation has been increasingly used in food, beverages, waste treatment, and functional products. In terms of soy sauce production, the yield of amino acid nitrogen in soy sauce fermented by multiple bacteria is significantly higher than that in soy sauce fermented by single bacteria; in terms of fruit wine brewing, compared with simple brewer's yeast fermentation, mixed bacteria fermentation has more types and contents of volatile substances in fruit wine, and the flavor is more coordinated; in terms of functional products, compared with single strains, mixed bacteria fermentation has synergistic effects between strains, faster growth rate, and higher enzyme activity. For example, a method for preparing mixed bacteria fermented fruit wine disclosed in patent application 202210259511.X is to mix 60-80 parts of fruit juice, 10-20 parts of fermented rice wine, and 2-5 parts of red yeast barley, ferment at 25-30°C for 7-10 days, and continue to ferment at 14-16°C for 50-60 days after pouring to obtain fermented fruit wine; the fruit juice includes but is not limited to plum juice, apple juice, orange juice, grape juice, and hawthorn juice. The obtained mixed bacteria fermented fruit wine can significantly increase the dry matter content in the wine, improve the quality of the fruit wine, and make the taste of the fruit wine richer and softer. Patent application 202211251108.9 discloses a method for preparing a mixed bacteria fermented tea lozenge, comprising the following steps: Step 1: Adding Aspergillus niger and Aspergillus niger to tea leaves for fermentation to obtain fermented tea leaves; Step 2: Mixing the fermented tea leaves with water in a mass ratio of 1:10-14, extracting, filtering and concentrating to obtain a mixed bacteria fermented tea extract; Step 3: Mixing the mixed bacteria fermented tea extract, hawthorn extract, mung bean extract, lily extract and ginger extract to obtain a mixed extract; Step 4: Evenly mix the xylitol aqueous solution, mixed extract, citric acid, monk fruit powder and mint powder, and then pour them into a tablet mold, and cool them to obtain mixed bacteria fermented tea lozenges. The obtained product has antioxidant capacity in vitro and in vivo, and has a certain ability to scavenge ABTS and DPPH free radicals.

[0004] However, with the development of mixed bacterial fermentation technology, common combined mixed bacterial culture, sequential mixed bacterial culture, etc. all require knowing the proportion and number of strains in the fermentation broth to adjust the conditions of the fermentation experiment. However, the counting method of individual strains in mixed bacterial fermentation broth is relatively complicated. At present, conventional methods such as dilution coating plates, microscopic observation and Gram staining can effectively count single bacterial fermentation strains, but because the morphology and size of strains in mixed bacterial fermentation are similar, the above methods cannot clearly calculate the number of single bacteria in the fermentation broth, resulting in inaccurate counting and consuming a lot of manpower and material resources. Summary of the invention

[0005] The present invention provides a fluorescent quantitative PCR primer and application thereof in probiotic bacteria counting in mixed bacteria fermentation, which can effectively solve the above problems.

[0006] The present invention is achieved in that:

[0007] A fluorescent quantitative PCR primer set, comprising fluorescent quantitative PCR primers for Staphylococcus xylosus, fluorescent quantitative PCR primers for Lactobacillus mucilaginosus fermentans and fluorescent quantitative PCR primers for Bacillus plantarum;

[0008] The upstream primer of the fluorescent quantitative PCR primers of Staphylococcus xylosus is shown in SEQ ID No.1, and the downstream primer is shown in SEQ ID No.2;

[0009] The upstream primer of the fluorescent quantitative PCR primers for the fermented mucus lactobacillus is shown in SEQ ID No.3, and the downstream primer is shown in SEQ ID No.4;

[0010] The upstream primer of the fluorescent quantitative PCR primer of Lactobacillus plantarum is shown in SEQ ID No.5, and the downstream primer is shown in SEQ ID No.6.

[0011] The invention discloses an application of the fluorescent quantitative PCR primer set in measuring the number of Staphylococcus xylosus, Lactobacillus mucilaginosus and Lactobacillus plantarum in mixed bacterial fermentation.

[0012] In some embodiments, the use of the fluorescent quantitative PCR primer set in measuring the number of Staphylococcus xylosus, Lactobacillus mucilaginosus, and Lactobacillus plantarum in mixed bacterial fermentation comprises the following steps:

[0013] S1, extracting DNA of single bacteria of Staphylococcus xylosus, Lactobacillus fermentans and Lactobacillus plantarum respectively, performing PCR amplification using the corresponding fluorescent quantitative PCR primers respectively, purifying the PCR amplified products, connecting the PCR amplified products with plasmid vectors, transferring them into competent cells, screening positive clones for PCR amplification, and obtaining plasmids of Staphylococcus xylosus, Lactobacillus fermentans and Lactobacillus plantarum;

[0014] S2, diluting the Staphylococcus xylosus plasmid, the fermented Lactobacillus mucilaginosus plasmid and the plantarum lactobacillus plasmid obtained in step S1 in a gradient manner as standard products, and performing a fluorescence quantitative PCR experiment using the corresponding fluorescence quantitative PCR primers; and then drawing a standard curve based on the Cq value obtained by the fluorescence quantitative PCR and the plasmid copy number; the plasmid copy number is calculated based on the plasmid concentration;

[0015] S3, respectively, single-bacterial fermentation of Staphylococcus xylosus, Lactobacillus fermentans and Lactobacillus plantarum, extracting DNA, performing fluorescent quantitative PCR amplification experiments with the corresponding fluorescent quantitative PCR primers, and calculating the plasmid copy number according to the standard curve drawn in step S2; at the same time, performing dilution plate coating counting to obtain the corresponding single-bacterial bacterial count CFU value; analyzing the obtained plasmid copy number and the corresponding single-bacterial bacterial count CFU value to obtain a linear relationship curve between the plasmid copy number and the single-bacterial bacterial count CFU value;

[0016] S4, extracting the total DNA of the mixed fermentation of Staphylococcus xylosus, Lactobacillus fermentans and Lactobacillus plantarum, using the corresponding fluorescent quantitative PCR primers, respectively performing fluorescent quantitative PCR amplification experiments, calculating the plasmid copy number according to the standard curve drawn according to step S2, and then calculating the strain number CFU value of each bacteria according to the linear relationship curve between the plasmid copy number and the single bacterial count CFU value obtained in step S3.

[0017] In some embodiments, the fluorescent quantitative PCR system is: TB Green Premix Ex Taq 10 μL, PCR Forward Primer 0.8 μL, PCR Reverse Primer 0.8 μL, ROX ReferenceDye II 0.08 μL, DNA template 2 μL, ddH 2 O 10.5 μL.

[0018] In some embodiments, the fluorescence quantitative PCR programs of Staphylococcus xylosus and Lactobacillus fermentans are set to: 94°C 30s; 40 cycles (denaturation, annealing, extension): 94°C 5s, 55°C 20s (fluorescence collection), 72°C 1min (fluorescence collection); melting, 95°C 15s, 60°C 1min, 95°C 1s (fluorescence collection).

[0019] In some embodiments, the fluorescence quantitative PCR program for Lactobacillus plantarum is set as: 94°C 30 s, 35 cycles (denaturation, annealing, extension): 94°C 5 s, 55°C 30 s (fluorescence collection), melting: 95°C 15 s, 60°C 1 min, 95°C 1 s (fluorescence collection).

[0020] A fluorescent quantitative PCR primer for Staphylococcus xylosus, wherein the upstream primer is shown as SEQ ID No.1, and the downstream primer is shown as SEQ ID No.2.

[0021] A fluorescent quantitative PCR primer for fermenting mucus lactobacillus, wherein the upstream primer is shown in SEQ ID No.3, and the downstream primer is shown in SEQ ID No.4.

[0022] A fluorescent quantitative PCR primer for Lactobacillus plantarum, wherein the upstream primer is shown as SEQ ID No.5, and the downstream primer is shown as SEQ ID No.6.

[0023] A fluorescence quantitative PCR kit comprises at least one of the fluorescence quantitative PCR primers for Staphylococcus xylosus, the fluorescence quantitative PCR primers for Lactobacillus mucilaginosus fermentans, and the fluorescence quantitative PCR primers for Lactobacillus plantarum.

[0024] The beneficial effects of the present invention are:

[0025] The specific primers of the present invention are designed according to the characteristic positions on the conserved DNA fragment in the bacterial 16S gene, shorten the primer amplification fragment, make the results obtained in the fluorescent quantitative PCR experiment more accurate, reduce the influence of the target fragment on the change of template integrity, can speed up the peak time, reduce the Cq value, and save time more conveniently.

[0026] The present invention extracts DNA of individual strains at different fermentation time periods and uses specific primers to perform fluorescence quantitative PCR experiments to obtain a standard curve of Cq value and copy number, and then constructs a related linear curve through the copy number and the CFU value of the strain obtained by dilution coating plates, performs DNA extraction and specific primer quantification on the mixed bacterial fermentation broth, and substitutes the curve to calculate the number of single strains, which can simply and quickly calculate the number of specific strains in the fermentation sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0028] Figure 1 This is a schematic diagram of the Cq value and copy number lg value obtained for the Staphylococcus xylosus plasmid in Example 2 of the present invention.

[0029] Figure 2 This is a schematic diagram of the Cq value and the copy number lg value obtained by fermenting the Lactobacillus mucilaginosus plasmid in Example 2 of the present invention.

[0030] Figure 3 The figure is a schematic diagram of the Cq value and the copy number lg value obtained by the Lactobacillus plantarum plasmid in Example 2 of the present invention.

[0031] Figure 4 Schematic diagram of gel electrophoresis bands after strain amplification using two different specific primers in Example 2 of the present invention.

[0032] Figure 5 Schematic diagram of the linear relationship between the copy number of Staphylococcus xylosus fluorescence quantitative PCR and the CFU value of the plate counting result in Example 3 of the present invention.

[0033] Figure 6 Schematic diagram of the linear relationship between the copy number of Lactobacillus fermentans by fluorescent quantitative PCR and the CFU value of the plate counting result in Example 3 of the present invention.

[0034] Figure 7 Schematic diagram of the linear relationship between the copy number of Lactobacillus plantarum fluorescent quantitative PCR and the CFU value of the plate counting result in Example 3 of the present invention. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the invention claimed for protection, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0036] The embodiment of the present invention aims to provide a primer combination for fluorescent quantitative PCR technology, which is particularly suitable for detecting and quantifying specific microbial species. Specifically, the primer combination includes fluorescent quantitative PCR primers for Staphylococcus xylosus, Limosilactobacillusfermentum and Lactiplantibacillus plantarum. These primers can specifically identify and amplify specific gene sequences of target microorganisms, thereby achieving accurate detection and quantitative analysis of these microorganisms.

[0037] In the present invention, the fluorescent quantitative PCR primers for Staphylococcus xylosus include an upstream primer and a downstream primer, whose sequences correspond to SEQ ID No. 1 and SEQ ID No. 2, respectively. These primers are designed based on a conserved region in the 16SrRNA gene of Staphylococcus xylosus, which is located at base positions 170-195 of the gene sequence. By selecting this specific conserved region as the detection target, it can be ensured that the primers have high specificity and sensitivity to Staphylococcus xylosus.

[0038] Similarly, the fluorescent quantitative PCR primers for fermentation mucus lactobacillus also include an upstream primer and a downstream primer, whose sequences correspond to SEQ ID No. 3 and SEQ ID No. 4, respectively. These primers are designed based on a conserved region in the 16S rRNA gene of fermentation mucus lactobacillus, which is located at base positions 172-200 of the gene sequence. The selection of this conserved region enables the primers to specifically recognize and amplify the DNA of fermentation mucus lactobacillus, thereby achieving accurate detection of the microorganism.

[0039] Finally, the fluorescent quantitative PCR primers for Lactobacillus plantarum also include an upstream primer and a downstream primer, whose sequences correspond to SEQ ID No. 5 and SEQ ID No. 6, respectively. These primers are designed based on a conserved region in the 16SrRNA gene of Lactobacillus plantarum, which is located at base positions 140-170 of the gene sequence. By selecting this specific conserved region as the detection target, it can be ensured that the primers have high specificity and sensitivity to Lactobacillus plantarum.

[0040] In summary, the fluorescent quantitative PCR primer combination provided by the present invention can be effectively used to detect and quantify Staphylococcus xylosus, Lactobacillus fermentans and Bacillus plantarum. The design of these primers is based on the conserved region in the 16SrRNA gene of the respective target microorganisms, ensuring the specificity and accuracy of the detection. By using these primers to carry out fluorescent quantitative PCR reaction, researchers and clinical diagnostic personnel can quickly and accurately identify and quantify the presence of these microorganisms, thereby having a wide range of application value in the fields of food testing, environmental monitoring and clinical diagnosis.

[0041] The embodiment of the present invention provides an application of the fluorescent quantitative PCR primer set in measuring the number of Staphylococcus xylosus, Lactobacillus fermentans, and Lactobacillus plantarum in mixed bacterial fermentation. Fluorescent quantitative PCR (Real-time PCR) is a method for quantitatively analyzing unknown templates through real-time detection of the fluorescent signal of each cycle product in the amplification reaction, and finally through a standard curve. The number of cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold is called the Cq value, which is linearly related to the logarithm of the starting concentration, and the value is reproducible, and the copy number of the target gene in the sample is determined by absolute quantification. Compared with the existing primers, the specific primers of the embodiment of the present invention can speed up the peak time, reduce the Cq value, save time more conveniently, and are applied to the counting of the number of strains in mixed bacterial fermentation, and the counting is accurate and reliable.

[0042] In some specific embodiments, the use of the fluorescent quantitative PCR primer set involved in the present invention in determining the number of Staphylococcus xylosus, Lactobacillus mucilaginosus and Lactobacillus plantarum in a mixed bacterial fermentation process comprises the following steps:

[0043] First, step S1 involves extracting DNA of single strains of xylosus Staphylococcus, fermented mucus Lactobacillus and plant lactobacillus respectively. Then, the corresponding fluorescent quantitative PCR primers provided by the present invention are used to perform PCR amplification on these DNAs. The amplified PCR product needs to be purified, and then the purified PCR product is connected to the plasmid vector. The connected product needs to be transferred into competent cells, and by screening positive clone colonies and performing PCR amplification, the xylosus Staphylococcus plasmid, the fermented mucus Lactobacillus plasmid and the plant lactobacillus plasmid are finally obtained.

[0044] Secondly, step S2 involves gradient dilution of the Staphylococcus xylosus plasmid, the fermented Lactobacillus mucilaginosus plasmid and the plant lactobacillus plasmid obtained in step S1 as standard products. The corresponding fluorescent quantitative PCR primers provided by the present invention are used to perform a fluorescent quantitative PCR experiment. The Cq value obtained by the experiment and the plasmid copy number are used to draw a standard curve. The plasmid copy number is calculated based on the plasmid concentration.

[0045] Next, step S3 involves single bacterial fermentation of Staphylococcus xylosus, Lactobacillus fermentans and Bacillus plantarum respectively. After fermentation is completed, DNA is extracted, and a fluorescent quantitative PCR amplification experiment is performed using the corresponding fluorescent quantitative PCR primers provided by the present invention. According to the standard curve drawn out in step S2, the plasmid copy number is calculated. At the same time, the dilution plate coating count is performed to obtain the corresponding single bacterial count CFU value. The obtained plasmid copy number is analyzed with the corresponding single bacterial count CFU value to obtain a linear relationship curve between the plasmid copy number and the single bacterial count CFU value.

[0046] Finally, step S4 involves extracting the total DNA of the mixed fermentation of Staphylococcus xylosus, Lactobacillus fermentans and Lactobacillus plantarum. Using the corresponding fluorescent quantitative PCR primers provided by the present invention, the fluorescent quantitative PCR amplification experiment is performed respectively. According to the standard curve drawn out in step S2, the plasmid copy number is calculated. According to the linear relationship curve between the plasmid copy number obtained in step S3 and the CFU value of the single bacterial amount, the strain number CFU value of each bacterium is calculated. By this method, the quantity of various bacteria in the mixed fermentation process can be accurately measured, thereby providing a scientific basis for the optimization and control of the fermentation process.

[0047] In some specific embodiments, the reaction system for fluorescent quantitative PCR includes the following components: 10 μL of TBGreen Premix Ex Taq, 0.8 μL of PCR Forward Primer, 0.8 μL of PCR Reverse Primer, 0.08 μL of ROX Reference Dye II, 2 μL of DNA template, and ddH 2O 10.5 μL. These components are precisely mixed together to ensure accuracy and reproducibility of the PCR reaction.

[0048] In some specific embodiments, the fluorescence quantitative PCR program for Staphylococcus xylosus and Lactobacillus fermentans is set to: first, a pre-denaturation step is performed at 94°C for 30 seconds; then 40 cycles are performed, each cycle including a denaturation step (at 94°C for 5 seconds), an annealing step (at 55°C for 20 seconds, and fluorescence signals are collected at this temperature) and an extension step (at 72°C for 1 minute, and fluorescence signals are collected at this temperature); finally, a melting curve analysis is performed, the steps of which include 15 seconds at 95°C, then 1 minute at 60°C, and finally 1 second of fluorescence signal collection at 95°C.

[0049] In some specific embodiments, the fluorescent quantitative PCR program for Lactobacillus plantarum is set to: first, a pre-denaturation step is performed at 94°C for 30 seconds; then 35 cycles are performed, each cycle including a denaturation step (at 94°C for 5 seconds), an annealing step (at 55°C for 30 seconds, and fluorescence signal collection is performed at this temperature); finally, a melting curve analysis is performed, the steps including at 95°C for 15 seconds, then at 60°C for 1 minute, and finally at 95°C for 1 second of fluorescence signal collection.

[0050] The embodiment of the present invention provides a fluorescent quantitative PCR primer for Staphylococcus xylosus, wherein the upstream primer is shown as SEQ ID No.1, and the downstream primer is shown as SEQ ID No.2.

[0051] The embodiment of the present invention provides a fluorescent quantitative PCR primer for fermenting Lactobacillus mucilaginosus, wherein the upstream primer is shown in SEQ ID No.3, and the downstream primer is shown in SEQ ID No.4.

[0052] A fluorescent quantitative PCR primer for Lactobacillus plantarum, wherein the upstream primer is shown as SEQ ID No.5, and the downstream primer is shown as SEQ ID No.6.

[0053] The embodiment of the present invention provides a fluorescent quantitative PCR kit, including at least one of the fluorescent quantitative PCR primers of xylosus Staphylococcus, the fluorescent quantitative PCR primers of fermented mucus Lactobacillus, and the fluorescent quantitative PCR primers of plant lactobacillus; the upstream primer of the fluorescent quantitative PCR primer of xylosus Staphylococcus is shown in SEQ ID No.1, and the downstream primer is shown in SEQ ID No.2; the upstream primer of the fluorescent quantitative PCR primer of fermented mucus Lactobacillus is shown in SEQ ID No.3, and the downstream primer is shown in SEQ ID No.4; the upstream primer of the fluorescent quantitative PCR primer of plant lactobacillus is shown in SEQ ID No.5, and the downstream primer is shown in SEQ ID No.6. The kit can be used for counting each bacterium in mixed bacterial fermentation, and can also be used for counting each single bacterium.

[0054] Example 1: Design of primers specific to three probiotic strains and construction of amplified fragment pUCm-T plasmid.

[0055] Three pairs of primers were constructed with 170-195 positions on the conserved DNA fragment in the 16S gene of Staphylococcus xylosus as detection targets, 172-200 positions on the conserved DNA fragment in the 16S gene of Lactobacillus plantarum as detection targets, and 140-170 positions on the conserved DNA fragment in the 16S gene of Lactobacillus fermentans as detection targets, as follows: Staphylococcus xylosus F (5'-TGCACATCTTGACGGTACCT-3') (SEQ ID No.1) and Staphylococcus xylosus R (5'-GTTTCCAATGACCCTCCACG-3') (SEQ ID No.2) (M); Lactobacillus fermentans F (5'-TACTCTGTGCACCTTAGGCG-3') (SEQ ID No.3) and Lactobacillus fermentans R (5'-GCTAGTAATCGCGGATCAGC-3') (SEQ ID No.4) (F); Lactobacillus plantarum F (5'-TGCACATCTTGACGGTACCT-3') (SEQ ID No.5) and Lactobacillus fermentans R (5'-GCTAGTAATCGCGGATCAGC-3') (SEQ ID No.6) (7); (5'-CGTTACCCCACCATCTAGCT-3')(SEQ ID No.5) and Lactobacillus plantarum R (5'-GTGGCGAACTGGTGAGTAAC-3')(SEQ IDNo.6) (Z). Among the three selected strain gene sequences, only the corresponding strain has a high degree of homology in the primer fragment, and has significant sequence differences with the other two strains. In addition, the three pairs of primers reduce the formation of primer dimers by base complementary pairing between primers, shorten the primer amplification fragment, make the results obtained in the fluorescent quantitative PCR experiment more accurate, and reduce the influence of changes in template integrity on the target fragment.

[0056] The three probiotic strains were Staphylococcus xylosus TG022 (preservation number: CCTCC NO: M 2023516, preservation date 2023-04-10, classified as Staphylococcus xylosus TG022), Lactobacillus fermentum TG017 (preservation number: CCTCC NO: M 2023513, preservation date 2023-04-10, classified as Limosilactobacillusfermentum TG017), and Lactobacillus plantarum TG008 (preservation number: CCTCC NO: M 20221458, preservation date 2022-09-20, classified as Lactiplantibacillus plantarum TG008) were activated on BHIS solid medium, and then single colonies were picked and expanded in BHIS liquid medium for 24 h (BHIS medium formula: tryptone 10.0 g / L; sodium chloride 5.0 g / L; sodium hydrogen phosphate 2.5 g / L; glucose 2.0 g / L; cattle heart extract 500.0 mL / L; yeast extract 5.0 g / L; L-cysteine ​​0.5 g / L; resazurin mother solution 10.0 mL / L; hemin mother solution 10.0 mL / L; vitamin K1 mother solution 100.0 μL / L; final pH 7.4 ± 0.2, sterilized at 120 ° C for 20 min). The DNA of the three strains was extracted using a bacterial genomic DNA extraction kit (Tiangen Biotechnology), and PCR amplification was performed using the above-mentioned specific primers. The above-mentioned strains are preserved in the China Center for Type Culture Collection, Wuhan, China.

[0057] In addition, existing primers were selected as control primers to perform PCR amplification under the same conditions. The existing primers are as follows: Staphylococcus xylosus F2 (5'-AACGCGCAACGTGATAAAATTAATG-3') (SEQ ID No.7) and Staphylococcus xylosus R2 (5'-AACGCGCAACAGCAATTACG-3') (SEQ ID No.8) (M2); Lactobacillus fermentans F2 (5'-ACGTATGAACAGTTACTCTCATACGT-3') (SEQ ID No.9) and Lactobacillus fermentans R2 (5'-CCTGATTGATTTGGTCGCCAAC-3') (SEQ ID No.10) (F2); Lactobacillus plantarum F2 (5'-TGATCCTGGCTCAGGACGAA-3') (SEQ ID No.11) and Lactobacillus plantarum R2 (5'-TGCAAGCACCAATCAATACCA-3') (SEQ ID No.12) (Z2).

[0058] The PCR systems are shown in Tables 1-1, 1-2 and 1-3.

[0059] Table 1-1 Staphylococcus xylosus PCR reaction system

[0060]

[0061] Table 1-2 PCR reaction system of fermented Lactobacillus mucilaginosus

[0062]

[0063] Table 1-3 PCR reaction system of Lactobacillus plantarum

[0064]

[0065] The PCR program settings for Staphylococcus xylosus were: 94°C for 3 min; 35 cycles (denaturation, annealing, extension): 94°C for 10 s, 55°C for 10 s, 72°C for 30 s; 72°C for 5 min.

[0066] The PCR program settings for Lactobacillus fermentans were: 94°C for 3 min; 35 cycles (denaturation, annealing, extension): 94°C for 10 s, 55°C for 10 s, 72°C for 30 s; 72°C for 5 min.

[0067] The PCR program settings for Lactobacillus plantarum were as follows: 94°C for 3 min; 30 cycles (denaturation, annealing, extension): 94°C for 10 s, 60°C for 10 s, 72°C for 10 s; 72°C for 5 min.

[0068] The PCR products were detected by electrophoresis. The PCR products were spotted in the gel. The DL 2000Maker was used as the standard band. The electrophoresis was performed at 150V for 20 minutes. The bands were observed by gel imaging instrument. After amplification with self-selected specific primers, Staphylococcus xylosus, Lactobacillus fermentans and Lactobacillus plantarum had obvious bright bands at around 100-300 bp. After amplification with existing control primers, there were obvious bright bands at around 500bp, 400bp and 100bp, respectively ( Figure 4 ). Rapid DNA gel purification kit (TRANS) was used for gel excision and recovery.

[0069] The gel-cutting recovery product was connected to the pUCm-T plasmid (T vector PCR product cloning kit, Sangon) vector using the PCR product rapid ligation kit (Sangon Biotechnology Co., Ltd.) to carry the target fragment. The reaction system is shown in Table 2.

[0070] Table 2 Fragment ligation reaction system

[0071]

[0072] After the reaction system was prepared, it was placed in water at 16°C overnight.

[0073] After thawing 6 tubes of 50 μL DH5α competent cells on ice, add the connection liquid (Staphylococcus xylosus), connection liquid (Lactobacillus fermentans), and connection liquid (Bacillus plantarum), mix gently, and let stand on ice for 30 minutes; then heat shock in a water bath for 90 seconds, and then place in an ice bath for 5 minutes; add 300 μL of LB liquid culture medium, and culture in a shaking incubator at 300 rpm and 37°C for 1 hour. During the culture period, apply 20 μL IPTG (100 mM) and 100 μL X-gal (20 mg / mL) to the LB solid culture medium containing amp, and place it on the front side for use. After the culture is completed, use a pipette to draw 50 μL of liquid culture medium and apply it to the above LB solid culture medium, place it in a 37°C incubator in the forward direction for 1 hour, and then invert it for overnight culture.

[0074] The positive clone colonies (white) were picked up with a toothpick by blue-white screening on the plate, and PCR amplification was performed with primers M13F (5'-TGTAAAACGACGGCCAGT-3') (SEQ ID No. 13) and M13R (3'-CAGGAAACAGCTATGACC-5') (SEQ ID No. 14). The reaction system is shown in Table 3.

[0075] Table 3 Colony PCR system

[0076]

[0077] Plasmid extraction: The positive white colonies were inoculated into LB liquid culture medium containing amp, and cultured overnight at 300 rpm and 37°C in a shaker; 2 mL of bacterial solution was centrifuged at 5000 rpm for 5 min to collect the bacteria; the plasmid was extracted using a high-purity plasmid DNA mini-extraction kit (TRANS). The plasmid was sequenced after extraction and found to contain the target fragment, indicating that the target fragment was successfully transferred into the plasmid. The plasmid concentration was measured by an ultraviolet spectrophotometer (Tiangen Biotechnology). Plasmid copy number = (plasmid concentration * 6.0221 * 10^23) / (plasmid length * 10^9 * plasmid molar mass).

[0078] Specific primers: The plasmid concentration of Staphylococcus xylosus was 201.11 ng / μL (copy number 4.22E+10), the plasmid concentration of Lactobacillus fermentans was 143.54 ng / μL (copy number 3.11E+10), and the plasmid concentration of Lactobacillus plantarum was 98.65 ng / μL (copy number 2.30E+10).

[0079] Control primers: Staphylococcus xylosus plasmid concentration was 182.07 ng / μL (copy number 3.82E+10), Lactobacillus fermentans plasmid concentration was 148.70 ng / μL (copy number 3.22E+10), and Lactobacillus plantarum plasmid concentration was 64.31 ng / μL (copy number 1.50E+10).

[0080] Example 2: Standard curve of plasmid fluorescence quantitative PCR Cq value and plasmid copy number and primer comparison.

[0081] The xylose Staphylococcus plasmid, fermented Lactobacillus mucilaginosus plasmid and plantarum Lactobacillus plasmid obtained in Example 1 were respectively 10- 1 -10- 8 The gradient dilution was used as the standard for fluorescence quantitative PCR experiment. A standard curve was drawn based on the Cq value obtained by plasmid fluorescence quantitative PCR and the plasmid copy number, as shown in Figure 1-3 shown.

[0082] The reaction system and reaction procedure are shown in Table 4.

[0083] Table 4-qPCR reaction system

[0084]

[0085] The qPCR program settings for Staphylococcus xylosus and Lactobacillus fermentans DNA (three-step method) were as follows: 94°C for 30s; 40 cycles (denaturation, annealing, extension): 94°C for 5s, 55°C for 20s (fluorescence collection), 72°C for 1min (fluorescence collection); melting, 95°C for 15s, 60°C for 1min, 95°C for 1s (fluorescence collection).

[0086] Lactobacillus plantarum DNA qPCR program settings (two-step method): 94°C 30 s, 35 cycles (denaturation, annealing, extension): 94°C 5 s, 55°C 30 s (fluorescence collection), melting: 95°C 15 s, 60°C 1 min, 95°C 1 s (fluorescence collection).

[0087] The Cq values ​​after QPCR amplification of plasmids at different concentrations using specific primers (M, F, Z) and control primers (M2, F2, Z2) are shown in Table 5. The standard curve of Staphylococcus xylosus plasmid with specific primers ( Figure 1 ): y = -2.9195x + 33.289, R 2 =0.9991; fermentation Lactobacillus mucilaginosus plasmid standard curve ( Figure 2 ): y = -4.4842x + 45.946, R 2=0.9993; Lactobacillus plantarum plasmid standard curve ( Figure 3 ): y = -2.7379x + 31.664, R 2 =0.9993. Standard curve of Staphylococcus xylosus plasmid with control primers: y=-3.2349x+36.573, R 2 =0.994; fermentation mucus lactobacillus plasmid standard curve: y =-4.172x+45.224, R 2 =0.999; Lactobacillus plantarum plasmid standard curve: y = -2.633x+31.82, R 2 =0.998.

[0088] It can be seen from this that the Cq value using the specific primers (M, F, Z) of the present invention is shortened compared to the control primers (M2, F2, Z2), and the standard deviation of the standard curve is closer to 1.

[0089] Repeatability tests were carried out using 6 dilutions of a single bacterial plasmid as a template. The results showed that the coefficient of variation (CV) of the Cq value of the M primer was 0.50% to 7.24%, the coefficient of variation (CV) of the Cq value of the F primer was 0.45% to 1.26%, and the coefficient of variation (CV) of the Cq value of the Z primer was 0.17% to 0.83% (see Tables 6-8).

[0090] After PCR amplification of the strain with 6 primers, the gel electrophoresis band pattern is as follows Figure 4 As shown, the bands of the three specific primers M, F, and Z are brighter and have no dragging, which means they have better amplification efficiency.

[0091] This result shows that the three specific primers M, F, and Z have good repeatability, more sensitive detection and accuracy, and can perform stable and reliable detection.

[0092] Table 5 Comparison of Cq values ​​of standard curves constructed with different primers

[0093]

[0094] Table 6 Evaluation results of repeatability and stability of fluorescence quantitative PCR with primer M of Staphylococcus xylosus

[0095]

[0096] Table 7 Repeatability and stability evaluation results of primer F fluorescence quantitative PCR of fermented Lactobacillus mucilaginosus

[0097]

[0098] Table 8 Repeatability and stability evaluation results of primer Z fluorescence quantitative PCR of Lactobacillus plantarum

[0099]

[0100] Example 3: DNA extraction and plate counting of single-strain and mixed-strain fermentations of three probiotics.

[0101] Take OD 600 The strain seed liquid with a value between 0.8 and 1.0 was inoculated into 30 mL BHIS liquid medium at 1% volume for culture, and there were 4 groups: Staphylococcus xylosus group (M), fermented Lactobacillus mucilaginosus group (F), Lactobacillus plantarum group (Z), and three probiotic mixed fermentation group (MIX). The fermentation liquid was plated for single bacterial liquid counts at 6, 12, 24, 30, 36, and 48 hours (n=3), and the plate count mean results are shown in Table 9.

[0102] Table 9 Count results of single-bacteria fermentation liquid dilution plate coating (n=3)

[0103]

[0104] Example 4: Correlation analysis between the DNA copy number of fluorescent quantitative PCR and the number of bacteria counted on the plate at different time periods of single-bacteria fermentation, and calculation of the number of single bacteria in mixed-bacteria fermentation broth

[0105] Single bacterial DNA of three probiotics in Example 3 at 6, 12, 24, 30, 36, and 48 h was extracted for fluorescence quantitative PCR amplification experiment. The reaction system is shown in Table 4, and the reaction procedure is as shown in Example 2. According to the standard curve drawn in step Example 2, the plasmid copy number was calculated, and the obtained plasmid copy number was analyzed with the corresponding single bacterial count CFU value obtained in Example 3 to obtain a linear relationship curve between the strain fluorescence PCR copy number and the single bacterial count CFU value. Figure 4 ): y = 7.786x-50.41, R 2 =0.995; Lactobacillus fermentans ( Figure 5 ): y = 5.631x-33.10, R 2 =0.992; Lactobacillus plantarum ( Figure 6 ): y = 2.066x-6.648, R 2 =0.993.

[0106] The DNA of mixed bacteria fermentation 24 and 48h in Example 3 was extracted, and a fluorescence quantitative PCR amplification experiment was performed using three specific primers. The reaction system is shown in Table 4, and the reaction procedure is shown in Example 2. According to the standard curve drawn in step Example 2, the plasmid copy number was calculated, and the obtained plasmid copy number was brought into the linear relationship curve to obtain the bacterial amount CFU value of each bacteria. The results of the mixed bacteria fermentation dilution plate dilution count are shown in Table 10. As can be seen from Table 10, the copy number obtained by fluorescence quantitative PCR is brought into the obtained curve, and the obtained strain number is almost the same as the total bacterial amount result of the fermentation liquid dilution plate.

[0107] Table 10 The bacterial count calculated by fluorescent quantitative PCR of mixed bacterial DNA and the total bacterial count obtained by diluting and plating the mixed bacterial fermentation broth.

[0108]

[0109] It can be seen from this that detecting the number of strains in mixed bacterial fermentation by fluorescent quantitative PCR can more conveniently and quickly judge the number and proportion of strains in the fermentation broth, and the counting is more accurate and reliable.

[0110] In the above experiment, a standard curve was constructed by using the Cq value and copy number obtained by fluorescent quantitative PCR. After the linear relationship between the copy number and the strain quantity CFU was confirmed by comparing with the plate counting result, the Cq value of the fermentation sample was put into the curve, which can quickly and easily calculate the number of specific strains in the fermentation sample.

[0111] Different fermentation strains were measured, and corresponding specific primers were used to perform fluorescence quantitative PCR experiments to complete the construction of the plasmid standard curve. After the linear relationship between the bacterial count and the copy number of the corresponding strain was obtained, the number of strains could also be calculated.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A fluorescent quantitative PCR primer set, characterized in that: Including fluorescent quantitative PCR primers of Staphylococcus xylosus, fluorescent quantitative PCR primers of Lactobacillus fermentans and fluorescent quantitative PCR primers of Bacillus plantarum; The upstream primer of the fluorescent quantitative PCR primers of Staphylococcus xylosus is shown in SEQ ID No.1, and the downstream primer is shown in SEQ ID No.2; The upstream primer of the fluorescent quantitative PCR primers for the fermented mucus lactobacillus is shown in SEQ ID No.3, and the downstream primer is shown in SEQ ID No.4; The upstream primer of the fluorescent quantitative PCR primer of Lactobacillus plantarum is shown as SEQ ID No.5, and the downstream primer is shown as SEQ ID No.

6.

2. Use of the fluorescent quantitative PCR primer set according to claim 1 in measuring the number of Staphylococcus xylosus, Lactobacillus mucilaginosus, and Lactobacillus plantarum in mixed bacterial fermentation.

3. The use according to claim 2, characterized in that: The following steps are involved: S1, extracting DNA of single bacteria of Staphylococcus xylosus, Lactobacillus fermentans and Lactobacillus plantarum respectively, performing PCR amplification using the corresponding fluorescent quantitative PCR primers described in claim 1 respectively, purifying the PCR amplified products, connecting the PCR amplified products with plasmid vectors, transferring them into competent cells, screening positive clones for PCR amplification, and obtaining plasmids of Staphylococcus xylosus, Lactobacillus fermentans and Lactobacillus plantarum; S2, diluting the Staphylococcus xylosus plasmid, the fermented Lactobacillus mucilaginosus plasmid and the plantarum lactobacillus plasmid obtained in step S1 in a gradient manner as standard products, and performing a fluorescent quantitative PCR experiment using the corresponding fluorescent quantitative PCR primers described in claim 1; and then drawing a standard curve based on the Cq value obtained by the fluorescent quantitative PCR and the plasmid copy number; the plasmid copy number is calculated based on the plasmid concentration; S3, respectively fermenting Staphylococcus xylosus, Lactobacillus fermentans and Bacillus plantarum, extracting DNA, performing a fluorescent quantitative PCR amplification experiment with the corresponding fluorescent quantitative PCR primers described in claim 1, and calculating the plasmid copy number according to the standard curve drawn in step S2; at the same time, performing dilution plate coating counting to obtain the corresponding single bacterial count CFU value; analyzing the obtained plasmid copy number and the corresponding single bacterial count CFU value to obtain a linear relationship curve between the plasmid copy number and the single bacterial count CFU value; S4, extracting the total DNA of the mixed fermentation of Staphylococcus xylosus, Lactobacillus fermentans and Lactobacillus plantarum, using the corresponding fluorescent quantitative PCR primers described in claim 1, respectively performing fluorescent quantitative PCR amplification experiments, calculating the plasmid copy number according to the standard curve drawn according to step S2, and then calculating the single bacterial count CFU value of each bacteria according to the linear relationship curve between the plasmid copy number and the single bacterial count CFU value obtained in step S3.

4. The use according to claim 3, characterized in that: The system of the fluorescent quantitative PCR is: TB Green Premix Ex Taq 10 μL, PCR Forward Primer 0.8 μL, PCR Reverse Primer 0.8 μL, ROX Reference Dye II 0.08 μL, DNA template 2 μL, ddH2O 10.5 μL.

5. The use according to claim 4, characterized in that: The fluorescence quantitative PCR programs for Staphylococcus xylosus and Lactobacillus fermentans were set as follows: 94°C for 30 s; 40 cycles: 94°C for 5 s, 55°C for 20 s, 72°C for 1 min; melting, 95°C for 15 s, 60°C for 1 min, 95°C for 1 s.

6. The use according to claim 4, characterized in that: The fluorescence quantitative PCR program of Lactobacillus plantarum was set as follows: 94°C for 30 s, 35 cycles: 94°C for 5 s, 55°C for 30 s, melting: 95°C for 15 s, 60°C for 1 min, 95°C for 1 s.

7. A fluorescent quantitative PCR primer for Staphylococcus xylosus, characterized in that: The upstream primer is shown as SEQ ID No.1, and the downstream primer is shown as SEQ ID No.

2.

8. A fluorescent quantitative PCR primer for fermenting mucus lactobacillus, characterized in that: The upstream primer is shown in SEQ ID No.3, and the downstream primer is shown in SEQ ID No.

4.

9. A fluorescent quantitative PCR primer for Lactobacillus plantarum, characterized in that: The upstream primer is shown as SEQ ID No.5, and the downstream primer is shown as SEQ ID No.

6.

10. A fluorescent quantitative PCR kit, characterized in that: Comprising at least one of the fluorescent quantitative PCR primers described in any one of claims 7 to 8.

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