PMA-qPCR method for accurately quantifying live bacteria of animal bifidobacterium subsp. Lactis for food

Abstract

The invention discloses a method for rapidly detecting animal bifidobacterium subsp. Lactis, and particularly relates to a group of specific primer groups for detecting the animal bifidobacterium subsp. Lactis, a qPCR (quantitative polymerase chain reaction) viable bacterium detection method combined with PMA (polymethyl acrylate) dye and application of the qPCR viable bacterium detection method. The invention provides application of a nucleotide sequence (SEQ ID NO: 1) in identification of bifidobacterium animalis subsp. Lactis strains. Specific PCR primers are shown as SEQ ID NO: 2 and SEQ ID NO: 3. The qPCR analysis is carried out by combining PMA dye with the primer group, so that the rapid and accurate quantitative detection of the animal bifidobacterium subsp. Lactis viable bacteria is realized. The method has the remarkable advantages of being high in detection sensitivity, high in specificity and short in consumed time, targeted viable bacteria quantitative detection can be conducted on the bifidobacterium animalis subsp. Lactis in the compound probiotic product, reliable technical support is provided for the quality control and detection department of the probiotic product, and the important industrial application value is achieved.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial testing, and in particular to a primer for identifying animal Bifidobacterium lactis subspecies for food and a PMA-qPCR detection method for quantifying specific live bacteria and an application thereof. Background Art

[0002] Bifidobacterium animalis subsp. lactis ( Bifidobacterium animalum subsp. milk ), is a Gram-positive, rod-shaped, obligate anaerobic bacterium that often exists alone or in pairs. This species produces lactic acid and acetic acid by fermenting carbohydrates, has the function of maintaining intestinal microecological balance and promoting health, and is widely used in probiotic foods and health products. B. lactis It can colonize well on the surface of the host's intestinal mucosa and has a high tolerance to gastric acid and bile, indicating that it has a strong ability to adapt to the gastrointestinal environment and has the potential to provide health benefits to the host. Studies have shown that B. milk It can not only inhibit gastrointestinal pathogens by producing antimicrobial substances and competing for mucosal adhesion sites, but also interact with immune cells, and may play an important role in enhancing host immune function. In addition, some studies have found that B. lactis It may reduce the incidence of respiratory tract infections and shorten the course of the disease. As one of the most widely studied and applied probiotics, B. lactis It has been included in my country's "List of Bacteria that Can Be Used in Food", and is also included in the EU QPS list and the US GRAS list. It has a long history of safe use and has significant market value in the food industry.

[0003] There is a global consensus on the health benefits of probiotics for the human body, but their functionality and safety are strain-specific and closely related to the number of live bacteria ingested. As compound probiotic products dominate the market, the production and application of their multi-strain formulas have brought new challenges to food quality and safety, efficacy evaluation and industry standardization. Accurate identification and quantitative detection of live bacteria in single and mixed background strain products are important technical means to ensure product quality and achieve consistency control of the production process. At present, the current effective identification and live bacteria quantitative detection technologies mostly use traditional plate culture methods, which are relatively simple to operate, but the inspection cycle is long and cannot meet the needs of simultaneous detection of the number of live bacteria of each species / strain in compound probiotic products. There is an urgent need to develop new probiotic accurate identification and live bacteria counting technologies to meet the growing demand for probiotic product testing, promote industry development and improve product safety and effectiveness.

[0004] With the rapid development of molecular biology, the emergence of emerging technologies based on molecular targets, such as PMA-qPCR technology, has made it possible to count live bacteria in a composite bacterial system. Propidium monoazide (PMA) is a dye that has gradually attracted attention to distinguish between live and dead bacteria. Its combination with qPCR technology (PMA-qPCR) makes up for the limitation that traditional qPCR technology cannot detect live bacteria. PMA dye can enter cells with damaged cell membranes, cross-link with intracellular DNA molecules during photolysis, form stable nitrogen-carbon bonds, and prevent the subsequent PCR from amplifying DNA molecules, thereby filtering out dead cells from the sample, analyzing active cells with intact cell membranes in the sample, and achieving the purpose of distinguishing live and dead bacteria. Based on the dual requirements of primer specificity and cell membrane integrity, PMA-qPCR technology can not only efficiently identify microorganisms, but also achieve quantitative analysis of targeted live bacteria. It has become one of the core technologies for quantitative detection of live bacteria in probiotic products. In the field of food strain detection, especially in the quality control of composite probiotic products, PMA-qPCR technology has a clear application prospect. Therefore, the present invention adopts PMA-qPCR technology to establish a method for accurate identification and quantitative detection of live bacteria at the "subspecies" level of Bifidobacterium animalis lactis subspecies in composite probiotic products, aiming to provide an efficient and accurate detection method to ensure the quality and efficacy of probiotic products. Summary of the invention

[0005] The object of the present invention is to provide a pair of Bifidobacterium lactis subsp. Bifidobacterium animal subsp. milk ) and the PMA dye-based mechanism for distinguishing between live and dead bacteria were combined to establish a PMA-qPCR technology platform to achieve specific and accurate identification of animal Bifidobacterium lactis subspecies and quantitative detection of live bacteria in compound probiotic products.

[0006] The present invention proposes the use of the nucleotide sequence shown in SEQ ID NO: 1 for identifying Bifidobacterium animalis subsp. lactis.

[0007] The present invention provides a method for identifying animal Bifidobacterium lactis subspecies, and the detection steps are as follows: S1. Extract genomic DNA of the strain to be tested; S2. Design primers for the nucleotide sequence shown in SEQ ID NO: 1, and the sequences are shown in SEQ ID NO: 2 and SEQ ID NO: 3; S3, using primers to perform PCR amplification on the genomic DNA to obtain an amplified product; S4. The length of the target amplification product is 184 bp. If the actual amplification product is inconsistent with the length of the target amplification product, the strain to be tested is identified as non-animal Bifidobacterium lactis subsp. S5. If the length of the actual amplification product is consistent with that of the target amplification product, the strain to be tested is identified as Bifidobacterium animalis subsp. lactis.

[0008] The present invention provides a method for detecting the number of live bacteria of animal Bifidobacterium lactis subsp. lactis by using the PMA-qPCR method, and the detection steps are as follows: S1, treating the sample to be tested with PMA dye; S2, extracting DNA from samples after PMA staining; S3, performing qPCR amplification on the sample using the above primer set; S4. Substitute the Ct value obtained by amplification into the standard curve regression equation constructed based on the standard substance to obtain the number of live bacteria of Bifidobacterium animalis subsp. lactis in the sample to be tested.

[0009] The mass concentration of PMA during the PMA staining is 50 μMol / L;

[0010] The dark incubation time for PMA staining was 5 min;

[0011] The exposure time for PMA staining was 15 min.

[0012] The technical solution provided by the embodiment of the present invention has the following advantages compared with the prior art:

[0013] The present invention designs specific PCR detection primers based on the conservative gene sequence of animal Bifidobacterium lactis subspecies, and the specific molecular marker can be used to accurately identify animal Bifidobacterium lactis subspecies in composite probiotic products without the need to separate single colonies; and the present invention establishes a PMA-qPCR technology platform, which can not only efficiently distinguish between dead and live bacteria, but also can quickly and accurately quantify the number of live bacteria of animal Bifidobacterium lactis subspecies in composite probiotics. Compared with traditional methods, PMA-qPCR technology has significant advantages in distinguishing between dead and live bacteria, improving detection sensitivity and accuracy, and can meet the high requirements of strain activity and quantity detection in complex probiotic systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1-Figure 5The electrophoretic diagrams of PCR specific amplification verification of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5, SEQ ID NO: 6 and SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively, wherein M-2k DNA marker; 1-animal Bifidobacterium lactis subsp. B . milk CICC 24210 T ; 2- Bifidobacterium animalis subsp. lactis B . milk CICC 21712; 3-Bifidobacterium animalis subsp. lactis B . milk CICC 21709; 4-Bifidobacterium animalis subsp. lactis B . milk CICC 21710; 5-Bifidobacterium animalis subsp. lactis B . milk CICC 6167; 6-Bifidobacterium animalis subsp. infantis B. infant CICC 6069 T ; 7- Bifidobacterium bifidum B. bifidum CICC 6071 T ; 8-Bifidobacterium breve B. short CICC 6079 T ; 9- Bifidobacterium adolescentis B. of a young man CICC 6070 T ; 10- Bifidobacterium animalis subsp. B. animal CICC 6250 T ; 11- Bifidobacterium longum subsp. longum B. longum CICC 6186 T ; CK-blank control.

[0016] Figure 6 This is the electrophoresis diagram of inclusive PCR amplification verification of primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, where M-2k DNA marker; P-positive control B . milk CICC 24210 T ; The rest are Bifidobacterium animalis subsp. lactis, 1- B . milk CICC 21714;2- B . milk CICC 10071R;3- B . milk CICC 10055R;4- B . milk CICC 10142R;5- B . milk CICC 10190R;6- B . milk CICC 10070R;7- B . milk CICC10014R;8- B . milk CICC 10071R;9- B . milk CICC 10137R;10- B . milk CICC 21712;11- B . milk CICC 10189R;12- B . milk CICC 10038R;13- B . milk CICC 6167;14- B . milk CICC 10039R;15- B . milk CICC 21709;16- B . milk CICC 24672;17- B . milk CICC 21710;18- B . milk CICC 10036R;19- B . milk CICC 10140R; CK-blank control.

[0017] Figure 7 This is the electrophoresis diagram of exclusive PCR amplification verification of the primers shown in SEQ ID NO: 2 and SEQ ID NO: 3, where M-2k DNA marker; P-positive control B . milk CICC 24210 T ; 1-Lactobacillus casei L. casei CICC 6117 T ; 2-Lactobacillus rhamnosus L. rhamnosus CICC 6224 T ; 3-Lactobacillus plantarum L. plantarum CICC 6240 T; 4-Lactobacillus reuteri L. reuteri CICC 6132 T ; 5-Lactobacillus mucilaginosus fermentation L. fermentum CICC 24209 T ; 6-Lactobacillus delbrueckii subsp. bulgaricus L. delbrueckii subsp. Bulgarian CICC 6013 T ; 7-Lactobacillus acidophilus L. acidophilus CICC 6081 T ; 8-Lactobacillus gasseri L. gasseri CICC 24878 T ; 9-Lactobacillus helveticus L. Swiss CICC 24208 T ; 10-Lactobacillus johnsonii L. johnsonii CICC 6252 T ; 11-Lactobacillus sakei L. sake CICC 6245 T ; 12- Bifidobacterium animalis subsp. B. animal CICC 6250 T ; 13- Bifidobacterium adolescentis B. of a young man CICC 6070 T ; 14-Bifidobacterium breve B. short CICC 6079 T ; 15- Bifidobacterium longum subsp. longum B. long CICC 6186 T ; 16- Bifidobacterium longum subsp. infantis B. infant CICC 6069 T ; 17- Bifidobacterium bifidum B. bifid CICC 6071 T ; 18- Streptococcus salivarius subsp. thermophilus S. thermophilus CICC 6222 T ; 19-Lactococcus lactis subsp. L. lactis CICC 6246 T ; 20-Lactococcus cremoris L. cremoris CICC 24337 T ; 21- Propionic acid producing bacteria P. acidipropionicus CICC 24923 T ; 22- Leuconostoc mesenteroides subsp. mesenteroides L. mesenteroides CICC25070 T ; 23-Bovine Zoococcus S. vitulinusCICC 10850 T ; 24-Lactobacillus crispatus Lucius Crispus JCM1185 T ; 25-Staphylococcus xylosus S. xylosus JCM 2418 T ; 26-Propionibacterium freudenreichii subsp. P. Sherman CGMCC 1.2231 T ;27- Pediococcus acidilactici P. acidilactici CGMCC 1.2696 T ; 28-Pediococcus pentosaceus P. pentosaceus CGMCC 1.2695 T ; 29-Weizmannella coagulans B. coagulans CGMCC 1.2009 T ; 30- Lactobacillus kumiss subsp. L.kefiranofaciens CGMCC 1.3402 T ; 31-Salivary Lactobacillus L. salivary CGMCC 1.1881 T ; 32-Staphylococcus carnosus S. carnosus ACCC 01657; 33-Lactobacillus curvatus L. curvatus JCM 1096 T ; 34-Lactobacillus delbrueckii subsp. lactis L. delbrueckii subsp. milk CGMCC1.2625 T ; 35-Lactococcus cremoris L. mesenteroides subsp. cream CICC 22181; CK-blank control.

[0018] Figure 8 It is the standard curve between the number of live bacteria of Bifidobacterium animalis subsp. lactis and the Ct value.

[0019] Figure 9 These are the results of the PMA-qPCR method's applicability test for different strains of Bifidobacterium lactis subsp. DETAILED DESCRIPTION

[0021] In order to more clearly understand the above-mentioned objectives, features and advantages of the present invention, the scheme of the present invention will be further described below. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

[0022] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein; it is obvious that the embodiments in the specification are only part of the embodiments of the present invention, rather than all of the embodiments.

[0023] The first aspect of the embodiments of the present invention provides an application of a conserved gene sequence of Bifidobacterium animalis subsp. lactis in identifying strains of Bifidobacterium animalis subsp. lactis, including specific primer design and PCR amplification to verify primer specificity.

[0024] The present invention uses CD-hit to predict the pan-genome, obtains the core gene set of animal Bifidobacterium lactis subspecies, and compares the core gene set with the genes of the same species and different subspecies strains of animal Bifidobacterium lactis subspecies in the NCBI database. The Pyridoxal phosphate homeostasisprotein gene of animal Bifidobacterium lactis subspecies with subspecies specificity and high conservatism is selected as the target sequence, and the nucleotide sequence is shown in SEQ ID NO:1.

[0025] When searching the NCBI NT database for SEQ ID NO:1, only 48 results of Bifidobacterium animalis subsp. lactis were obtained with a sequence similarity greater than 90% and a sequence coverage equal to 100%. The sequence similarity of the remaining subspecies of Bifidobacterium animalis was less than 80%. Therefore, SEQ ID NO:1 has a certain subspecies specificity for Bifidobacterium animalis subsp. lactis. Primers were designed using Prime Express software V3.0 (ABI, Foster City, CA, USA). Detailed information is shown in Table 1.

[0026] Table 1: Primer information of Bifidobacterium animalis subsp. lactis

[0027]

[0028] The genomic DNA of the strain to be tested was extracted, and PCR amplification was performed using the above primer set. The actual size of the amplified product was determined by gel electrophoresis imaging.

[0029] The length of the target amplification product is 184 bp. If the actual amplification product is inconsistent with the length of the target amplification product, the strain to be tested is identified as non-animal Bifidobacterium lactis subsp.

[0030] If the actual amplification product is consistent with the target amplification product in length, the strain to be tested is identified as Bifidobacterium animalis subsp. lactis.

[0031] The amplification system of the present invention is 50 μL, including 2 μL of DNA template, 2 μL×2 of upstream and downstream primers (10 μmol / L), 25 μL of 2×PCR Taqmix, and 19 μL of ddH2O; the PCR amplification conditions are 95°C for 5 min; 95°C for 30 s, 60°C for 34 s, and 72°C for 35 s, for a total of 35 cycles; and extension at 72°C for 10 min.

[0032] The second aspect of the embodiment of the present invention provides a PMA-qPCR method for quantitatively detecting live bacteria of Bifidobacterium animalis subsp. lactis in a composite probiotic product, and the steps are as follows.

[0033] 1) Sample pretreatment

[0034] Prepare the sample to be tested into a 1:10 sample solution, aseptically pipette 200 μL of the sample solution into a 96-well cell culture plate, and use an ELISA reader to measure the sample OD 620 The value is adjusted to 0.3~0.5, at which time the total number of bacteria is about 10 8 After adjusting the OD value, use a pipette to draw 500 μL of the solution and inject it into a 1.5 mL sterile centrifuge tube.

[0035] 2) Sample PMA treatment

[0036] Pipette 1.25 μL of 20 mmol / L PMA solution and inject it into 500 μL sample solution to make the final PMA concentration 50 μM / L. Incubate in the dark for 5 min, mix once every 1 min, and expose to LED light sensor (Biotium PT-H18A, USA) for 15 min. Centrifuge at 12000 rpm and room temperature for 10 min to collect the cells. Resuspend the cells in 200 μL sterile ddH2O for subsequent DNA extraction.

[0037] 3) Genomic DNA extraction

[0038] The bacterial suspension was pipetted into a 2 mL sterile grinding tube, into which 0.25 g sterile glass beads with a particle size of 0.1 mm were pre-loaded. A bead sample grinder was used, with a grinding speed of 6 m / s and a time of 12 s to break the bacterial cells. After centrifugation, the supernatant was pipetted as a crude DNA extract.

[0039] 4) Real-time fluorescence quantitative PCR detection

[0040] qPCR reaction system: SYBR qPCR Mix 10 μL, upstream and downstream primers 10 μmol / L 0.4 μL each, 50×ROX 0.08 μL, diluted DNA template 2 μL, add sterile ultrapure water to make up the volume to 20 μL, and use ABI 7500 real-time fluorescence quantitative PCR instrument for amplification. qPCR reaction conditions: 95℃ / 30 s, 1 cycle; 95℃ / 5 s, 60℃ / 34 s (collect fluorescence), a total of 40 cycles.

[0041] 5) Preparation of standard curve

[0042] Bifidobacterium animalis subsp. lactis was inoculated on MRS solid medium and cultured anaerobically at 37°C for 48 h. An appropriate amount of bacteria was selected and resuspended in sterile saline. The concentration of the bacterial solution was adjusted to about 10 8 CFU / mL, the plate count method was used to detect the number of viable bacteria (CFU / mL). 500 μL of bacterial suspension was aspirated, treated with the above PMA conditions, and genomic DNA was extracted using a bead sample grinder. After ten-fold gradient dilution of genomic DNA, qPCR amplification was performed to establish the relationship between qPCR Ct value and viable bacterial colony concentration (Log 10 CFU / mL), through the formula E=10 -1 / s -1 to calculate the amplification efficiency, where s is the slope of the standard curve.

[0043] The standard curve is as follows Figure 8 As shown in the figure, the linear regression equation is y=-3.432x+41.894, where y represents the qPCR amplification Ct value and x represents the logarithmic value of the total number of viable bacteria (Log 10 CFU / mL). The amplification efficiency of the standard curve was 95.62%, which was in line with the theoretical range of 90%-110%, and the amplification efficiency was good; the correlation coefficient of the standard curve was R 2 =0.999, indicating that the qPCR amplification Ct value is proportional to the logarithm of the number of viable bacteria (Log 10 There was a good linear relationship between the

[0044] 6) Result analysis and calculation

[0045] For an unknown sample, blank control, negative control and positive control need to be set up during the test. The Ct value of the blank control and negative control should be greater than 30, and the Ct value of the positive control should be less than 25. The Ct value of the sample to be tested should be within the range of the standard curve. If it is not within the range of the standard curve, the sample DNA concentration needs to be appropriately adjusted and retested. For unknown samples that meet the above requirements, their Ct values ​​can be substituted into the standard curve, and the number of live bacteria of animal Bifidobacterium lactis subspecies contained therein can be obtained after conversion by the equation.

[0046] The benefit of the present invention is that accurate identification and live bacteria quantification of animal Bifidobacterium lactis subspecies in composite probiotic products are achieved through the PMA-qPCR method. The method has the advantages of high sensitivity, rapid detection, strong specificity, etc., and can efficiently and accurately perform targeted detection of animal Bifidobacterium lactis subspecies, and can effectively distinguish live bacteria from dead bacteria. PMA-qPCR technology has significant advantages in improving detection accuracy and efficiency, and provides reliable technical support for quality control, efficacy evaluation and consistency monitoring of composite probiotic products during production.

[0047] The embodiments of the present invention are further described below through specific examples. The experimental reagents used in the following examples are all commercially available, and the strains used are all existing strains preserved in the China Industrial Microbiological Culture Collection Center (CICC).

[0048] Example 1

[0049] This example is used to illustrate the primer design and specificity verification process.

[0050] 1) Primer design

[0051] Primers were designed using Prime Express software V3.0 for the core gene sequence of Bifidobacterium animalis subsp. lactis, and five pairs of specific primers were designed through a comprehensive evaluation of the primers using Oligo 7, as shown in Table 2. On this basis, experiments were conducted to verify the specificity of the primers.

[0052] Table 2: Primer information of Bifidobacterium animalis subsp. lactis

[0053]

[0054] 2) Primer specificity verification

[0055] Five strains of Bifidobacterium animalis subspecies lactis and six strains of the same species but different subspecies were selected, and the detailed information of the strains is shown in Table 3. After extracting the genomic DNA of the strains, PCR amplification was performed using the primer sets shown in Table 2 to verify the specificity and effectiveness of the selected primer sets.

[0056] Table 3: PCR experiment verification strain information

[0057]

[0058] The PCR reaction system was 50 μL in total volume: 2 μL DNA template, 2 μL primers (10 μmol / L) × 2, 2×PCRTaqmix 25 μL, 19 μL ddH2O; PCR amplification conditions were 95°C for 5 min; 95°C for 30 s, 60°C for 34 s, 72°C for 35 s, 35 cycles; 72°C for 10 min. The amplified products were subjected to gel electrophoresis. The amplification results of the primers shown in SEQ ID NO:2 and SEQ ID NO:3 are shown in Figure 2. Figure 1 As shown, the amplification results of the primers shown in SEQ ID NO:4 and SEQ ID NO:5 are as shown Figure 2 As shown, the amplification results of the primers shown in SEQ ID NO: 6 and SEQ ID NO: 7 are as shown Figure 3 As shown, the amplification results of the primers shown in SEQ ID NO:8 and SEQ ID NO:9 are as shown Figure 4 As shown, the amplification results of the primers shown in SEQ ID NO: 10 and SEQ ID NO: 11 are as shown Figure 5 shown.

[0059] like Figure 1~Figure 5 The experimental results shown show that the primers shown in SEQ ID NO:2 and SEQ ID NO:3 can effectively amplify animal Bifidobacterium lactis subspecies, and no positive amplification is produced for non-animal Bifidobacterium lactis subspecies, and other primer sequences produce non-specific amplification for non-animal Bifidobacterium lactis subspecies. The above results show that the primers of SEQ ID NO:2 and SEQ ID NO:3 have strong specificity for animal Bifidobacterium lactis subspecies. In the follow-up, strains of different taxonomic status will be further collected to conduct a larger-scale verification of the primer set.

[0060] Example 2

[0061] This example is used to illustrate the inclusiveness of SEQ ID NO: 2 and SEQ ID NO: 3 primers to target strains and their exclusivity to non-target strains, that is, the ability to accurately detect target strains.

[0062] 20 strains of animal Bifidobacterium lactis subsp. were selected as experimental subjects, and the detailed information of the strains is shown in Table 4. After extracting the genomic DNA of the strains, PCR amplification was performed using primers SEQ ID NO: 2 and SEQ ID NO: 3, and the primers' tolerance to animal Bifidobacterium lactis subsp. was determined by gel electrophoresis. The PCR amplification conditions were the same as those in Example 1.

[0063] like Figure 6As shown, the primer sets SEQ ID NO: 2 and SEQ ID NO: 3 were positive for the PCR amplification products of 20 strains of Bifidobacterium animalis subspecies lactis, with a single band and a product size consistent with the expected 184 bp, indicating that the primers showed good specificity between different strains of Bifidobacterium animalis subspecies lactis.

[0064] Table 4: Primer inclusion experiment verification strain information

[0065]

[0066] 35 strains of the "List of Bacteria that can be used in food" were selected as experimental subjects, and 1 strain of animal Bifidobacterium lactis subspecies was selected as a positive control. The detailed information of the strains is shown in Table 5. After extracting the genomic DNA of the strains, PCR amplification was performed using primers of SEQ ID NO: 2 and SEQ ID NO: 3, and the exclusivity of the primers to non-animal Bifidobacterium lactis subspecies was determined by gel electrophoresis. The PCR amplification conditions were the same as in Example 1.

[0067] like Figure 7 As shown, the primer sets SEQ ID NO:2 and SEQ ID NO:3 were negative for the amplification products of 35 non-animal Bifidobacterium lactis subspecies, indicating that the primers can effectively distinguish between species within the genus, between genera within the family, and other strains. The inclusive and exclusive results showed that the primer sets SEQ ID NO:2 and SEQ ID NO:3 can distinguish the target strains from other non-target strains, have good specificity for animal Bifidobacterium lactis subspecies, and can achieve accurate identification of animal Bifidobacterium lactis subspecies in the composite strain system.

[0068] Table 5: Primer exclusivity verification experiment strain information

[0069]

[0070] Example 3

[0071] This example is used to illustrate that the PMA treatment conditions adopted can achieve effective differentiation between live and dead bacteria of Bifidobacterium animalis subsp. lactis.

[0072] 1) PMA treatment

[0073] Twenty strains of animal Bifidobacterium lactis subsp. lactis were selected and inoculated into MRS solid medium and cultured anaerobically at 37°C for 48 h. An appropriate amount of bacteria was selected and resuspended in sterile saline. The concentration of the bacterial solution was adjusted to about 10 8CFU / mL. The bacterial suspension was divided into two parts, one of which was a live bacterial suspension, and the other was treated in an 80℃ water bath for 15 min to prepare a dead bacterial suspension. 500 μL of the bacterial suspension was respectively injected into a sterile centrifuge tube, 1.25 μL of a 20 mmol / L PMA solution was injected into 500 μL of the sample solution to make the final PMA concentration of 50 μM / L, incubated in the dark for 5 min, mixed every 1 min, and exposed to an LED photosensitive instrument for 15 min. Centrifuged at 12000 rpm at room temperature for 10 min, collected the cells, and used for genomic DNA extraction. For the live and dead bacterial suspensions, a PMA-untreated group was set as a control.

[0074] 2) Genomic DNA extraction

[0075] Weigh 0.25 g of glass beads with a particle size of 0.1 mm, place them in a 2 mL crushing tube, and sterilize them at 121°C for 30 min. Use 200 μL of sterile ddH2O to resuspend the above bacteria, and transfer all the bacterial liquid to the crushing tube. Use a bead sample grinder at 6 m / s for 12 s to collect the crude DNA extract of the bacteria.

[0076] 3) Real-time fluorescence quantitative PCR detection

[0077] qPCR reaction system: SYBR qPCR Mix 10 μL, upstream and downstream primers 10 μmol / L 0.4 μL each, 50×ROX 0.08 μL, diluted DNA template 2 μL, add sterile ultrapure water to make up the volume to 20 μL, and use ABI 7500 real-time quantitative PCR amplification instrument for qPCR amplification. qPCR reaction conditions: 95℃ / 30 s, 1 cycle; 95℃ / 5 s, 60℃ / 34 s (collect fluorescence), a total of 40 cycles.

[0078] 4) Result analysis

[0079] The results were analyzed by inhibition rate (%) to ensure that the PMA action conditions could effectively inhibit the subsequent qPCR amplification of dead bacteria, while having no significant effect on the qPCR amplification of live bacteria, that is, effectively distinguishing between live and dead bacteria.

[0080] Inhibition rate (%) = (1-C 死菌-PMA(+) / C 死菌-PMA（-） )%.

[0081] C 死菌-PMA(+) : Bacterial concentration of the sample to be tested after PMA treatment (CFU / mL);

[0082] C 死菌-PMA（-） : Bacterial concentration of the sample without PMA treatment (CFU / mL).

[0083] The inhibition rate of PMA on dead bacteria qPCR is shown in Table 6. The inhibition rate of the established PMA-qPCR method on 20 strains of animal Bifidobacterium lactis dead bacteria reached 100.00%. In the table, PMA (-) means that the sample has not been treated with PMA; PMA (+) means that the sample has been treated with PMA.

[0084] Table 6: Inhibition rate of qPCR of dead bacteria under PMA conditions

[0085]

[0086] The results of qPCR detection of live bacteria under PMA conditions are shown in Table 7. The experimental results showed that there was no significant difference between the Ct values ​​of live bacteria after PMA treatment and those without treatment ( P >0.05). In the table, PMA (-) means the sample was not treated with PMA; PMA (+) means the sample was treated with PMA.

[0087] Table 7: PMA conditions for live bacteria qPCR detection results

[0088]

[0089] Example 4

[0090] This example is used to illustrate the applicability of the established PMA-qPCR method between different strains of Bifidobacterium animalis subsp. lactis.

[0091] 1) PMA treatment

[0092] Twenty strains of animal Bifidobacterium lactis subsp. lactis were selected and inoculated into MRS solid medium and cultured anaerobically at 37°C for 48 h. An appropriate amount of bacteria were selected and resuspended in sterile saline. The concentration of the bacterial solution was adjusted to about 10 8 CFU / mL. Pipette 500 μL of bacterial suspension into a sterile centrifuge tube, pipette 1.25 μL of 20 mmol / L PMA solution into 500 μL sample solution to make the final PMA concentration 50 μM / L, incubate in the dark for 5 min, mix once every 1 min, and expose to LED light sensor for 15 min. Centrifuge at 12000 rpm for 10 min at room temperature, collect the bacteria, and use them for genomic DNA extraction.

[0093] 2) Genomic DNA extraction

[0094] Same as Example 3.

[0095] 3) Real-time fluorescence quantitative PCR detection

[0096] Same as Example 3.

[0097] 4) Test results

[0098] After the sample DNA is amplified by qPCR, the Ct value is obtained and substituted into Figure 8 The standard curve shown in the figure (y=-3.432x+41.894) was converted to obtain the number of live bacteria of Bifidobacterium animalis subspecies lactis in the sample. The PMA-qPCR live bacteria counts of different Bifidobacterium animalis subspecies lactis were compared with the plate counting results. T Test analysis. The results show that ( Figure 9 ), there was no significant difference between the two ( P >0.05), indicating that the established PMA-qPCR method can achieve accurate quantification of live bacteria of different strains of animal Bifidobacterium lactis subspecies and has good applicability among strains within subspecies.

[0099] Example 5

[0100] This example is used to illustrate the applicability of the established PMA-qPCR method for quantifying live bacteria of Bifidobacterium animalis subsp. lactis in products.

[0101] 1) Test sample information

[0102] Three commercially available probiotic products, A, B, and C, were purchased for method applicability study.

[0103] The labeled bacterial species of probiotic product A are: Lactobacillus rhamnosus, Bifidobacterium animalis subsp. lactis, Lactobacillus plantarum, Streptococcus salivarius subsp. thermophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus formatus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium breve, Bifidobacterium longum subsp. longum, and Bifidobacterium longum subsp. infantis. The labeled live bacterial count of Bifidobacterium animalis subsp. lactis is 6.00×10 9 CFU / g.

[0104] The bacterial species of probiotic product B are: Lactobacillus rhamnosus, Bifidobacterium breve, Lactobacillus plantarum, Bifidobacterium longum subsp. longum, Lactobacillus formatus, Streptococcus thermophilus, Bifidobacterium longum subsp. infantis, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium animalis subsp. lactis, and Lactobacillus reuteri. The number of live bacteria of Bifidobacterium animalis subsp. lactis is 2.73×10 10 CFU / g.

[0105] The probiotic product C contains the following bacterial species: Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus casei, Streptococcus thermophilus, Lactobacillus paracasei, Bifidobacterium animalis subsp. lactis, Lactobacillus gestatum, Lactobacillus reuteri, and Lactobacillus delbrueckii subsp. bulgaricus. The number of live bacteria in the Bifidobacterium animalis subsp. lactis subsp. lactis is 2.35×10 9 CFU / g.

[0106] 2) PMA treatment

[0107] Take 25 g of probiotic product and add it to 225 mL of sterile saline to fully dissolve and mix the sample. Use an ELISA reader to measure the OD value of the sample. 620 The value is adjusted to the range of 0.3~0.5, and the total number of live bacteria in the corresponding system is 10 8 CFU / mL. Take 500 μL of bacterial solution and add it to a 1.5 mL sterile centrifuge tube. Centrifuge at 12000 r / min for 10 min, discard the supernatant, and collect the bacteria. Take another 500 μL of saline to wash the bacteria once, then aspirate 500 μL of saline to resuspend the bacteria, add PMA solution to make the final concentration 50 μM, incubate in the dark for 5 min, mix every 1 min, and expose to LED photosensitizer for 15 min. Centrifuge at 12000 r / min at room temperature for 10 min, collect the bacteria, and use it for genomic DNA extraction.

[0108] 3) DNA extraction

[0109] Same as Example 3.

[0110] 4) Fluorescence quantitative PCR detection

[0111] Same as Example 3. The number of live bacteria of Bifidobacterium animalis subsp. lactis in the product was quantitatively detected by qPCR and compared with the claimed amount of bacteria added in the product.

[0112] 5) Test results

[0113] After the sample DNA is amplified by qPCR, the Ct value is obtained and the Ct value is substituted into Figure 8 In the standard curve shown (y=-3.432x+41.894), the number of live bacteria of Bifidobacterium animalis subsp. lactis in the sample was obtained by conversion. The number of live bacteria detected by PMA-qPCR method for samples A, B, and C was in the same order of magnitude as the claimed amount of the product, and the difference between the two was ± 0.5 Log 10 Within the range of CFU / g, the measured value is highly consistent with the claimed amount, indicating the accuracy of the detection results of the PMA-qPCR method. Products A, B, and C are all composed of multiple bacterial species, and they also have different subspecies and closely related species within the species of Bifidobacterium. The measured value is highly consistent with the claimed amount, which also shows that the designed animal Bifidobacterium lactis subspecies primers are highly specific and can accurately identify the target species in the compound probiotic products, achieve accurate quantification of targeted live microorganisms, and provide technical support for the quality assessment and market supervision of compound probiotic products.

[0114] Table 8: Comparison of PMA-qPCR test values ​​and product claims

[0115] .

Claims

1. A specific primer for identifying animal Bifidobacterium lactis subspecies, characterized in that, It includes a forward primer nucleotide sequence as shown in SEQ ID NO:2, and a reverse primer nucleotide sequence as shown in SEQ ID NO:

3.

2. A method for detecting the number of live bacteria of animal Bifidobacterium lactis subspecies for food, the method using the specific detection primer set according to claim 1, detecting animal Bifidobacterium lactis subspecies by PMA-qPCR, characterized in that, The method comprises the following steps: 1) PMA dye treatment; 2) genomic DNA extraction; 3) qPCR amplification using the primer set described in claim 1; 4) substituting the obtained Ct value into a standard curve constructed based on a standard substance to obtain the number of live bacteria of animal Bifidobacterium lactis subspecies.

3. The method according to claim 2, characterized in that In step 1), the PMA dye treatment parameters are as follows: the concentration of the bacterial solution in the sample to be tested is adjusted to OD 620 The concentration of PMA dye was about 0.3~0.

5. PMA dye was added to 500 μL of the solution to be tested. The final concentration of PMA dye was 50 μmol / L. After mixing, the mixture was reacted for 5 min in the dark. After exposure to an LED photosensitizer for 15 min, the bacteria were collected for subsequent experiments.

4. The method according to claim 2, characterized in that: In the step 3), the qPCR amplification system is as follows: 10 μL of SYBR qPCR Mix, 0.4 μL of each of the upstream and downstream primers at 10 μmol / L, 0.08 μL of 50×Rox, 2 μL of DNA template, and sterile ultrapure water is added to make up the volume to 20 μL; the qPCR amplification program is as follows: pre-denaturation at 95°C for 30 s, 1 cycle; denaturation at 95°C for 5 s, annealing / extension at 60°C for 34 s (collecting fluorescence), for a total of 40 cycles.

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