CN113564272B - Rapid identification and detection method for cheese lactobacillus in fermented milk - Google Patents
Rapid identification and detection method for cheese lactobacillus in fermented milk Download PDFInfo
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Abstract
The invention discloses a cheese lactobacillus (Lactobacillus casei) used in compound lactobacillus fermented milk and microbial inoculumLacticaseibacillus casei) And (3) rapidly identifying and detecting the method. Single copyrsmEThe gene nucleotide sequence is shown as SEQ ID No.1, and the specific PCR detection primers are shown as SEQ ID No.2 and SEQ ID No. 3. By using saidrsmEA specific PCR detection primer designed by a gene nucleotide sequence adopts a rapid identification and quantitative detection technology of azide propidium bromide-real-time fluorescence quantitative PCR (PMA-qPCR), PMA is added to effectively inhibit amplification of dead bacteria DNA, live non-culturable state (VBNC) bacteria which cannot be detected by a traditional method can be detected, and the Lactobacillus casei in the compound lactobacillus fermented milk and the microbial inoculum can be effectively identified and quantitatively detected. The detection method has the advantages of rapidness, accuracy and the like, and is convenient for dairy food agent enterprises and institutions such as three-party detection and the like to use.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a PMA-qPCR detection primer and a rapid detection method for specificity of lactobacillus casei in compound lactobacillus fermented milk and microbial inoculum products.
Background
Lactobacillus casei (L.casei) (II)Lacticaseibacillus casei) The microbial inoculum is widely existed in fermented foods such as cheese, fermented milk, pickle and the like and oral cavities and intestinal tracts of human bodies, is facultative anaerobic, does not move and produce spores, has the effects of regulating immunity, relieving intestinal inflammation, balancing intestinal flora, relieving constipation and the like, has long safety use history, and is listed in ' available food strain list ' and European Union safety Qualification (QPS) list ' in China. In 2020, Lactobacillus casei (L.casei)Lactobacillus casei) Taxonomical transition to Lactobacillus casei (Lacticaseibacillus casei) The national regulation standard has not been updated inCan be used in food strain list and fermented milk product identification and still be expressed as lactobacillus casei. Studies show that Lactobacillus casei: (Lacticaseibacillus casei) The functional and safety characteristics of probiotic bacteria are specific at the strain level. At present, a plurality of commercial strains are widely applied in the fields of fermented milk, microbial inoculum, solid beverage, health food and the like, and have great difference in the aspects of functionality, safety and the like.
The dairy industry is an important industry of healthy China and strong nationalities. Aiming at the current situations of weak research foundation, lagged standard regulation and law, monopoly of foreign technologies and the like in the dairy industry in China, the research target of the invention is to overcome the technical difficulties of inaccurate detection and unpleasant detection of the compound lactobacillus fermented milk and the microbial inoculum, namely the existing compound lactobacillus fermented milk and the microbial inoculum lactobacillus casei (lactobacillus casei)Lacticaseibacillus casei) The identification detection method mainly combines selective culture medium counting with physiological and biochemical detection and the like, the selective culture medium counting with physiological and biochemical detection and the like have complicated operation steps, operators need to have certain experience and skill, generally, the identification detection of one bacterium needs about 15 days, the time is very long, and the selection specificity and the identification accuracy are not high. The identification and detection methods such as Random Amplified Polymorphic DNA (RAPD), Pulse Field Gel Electrophoresis (PFGE), ribosome typing (Ribotyping) and the like need to use a pure culture sample as an analysis premise, and are not suitable for the compound lactobacillus fermented milk and microbial inoculum product samples.
The invention adopts PMA-qPCR specific detection technology to establish the composite lactobacillus fermented milk and the cheese bacillus in the microbial inoculumLacticaseibacillus casei) A method for precisely identifying and quantitatively detecting species level viable bacteria in a targeted manner. Through optimizing the condition of PMA treatment on the compound lactobacillus fermented milk sample, PMA is combined with DNA in dead cells to reduce PCR signals to obtain genome DNA treated by PMA, specific amplification primers and probe sequences of a species to be detected are designed, qPCR amplification is utilized to detect whether a specific amplification curve is presented or not, and the problem of target active lactobacillus casei (Lactobacillus casei) in the compound lactobacillus fermented milk and microbial inoculum is solvedLacticaseibacillus casei) Specific detection and quantification.
In recent years, the identification or quantitative determination of cheese bacillus by using related techniques in domestic and foreign literature or patent techniques is described below.
The invention discloses a method for detecting lactobacillus casei in probiotic dairy products, which is published in the patent publication No. CN 101153316B of the people's republic of China and has a patent name [ a method for detecting lactobacillus casei in probiotic dairy products ], and the method separates lactobacillus casei by using a culture medium and a culture medium added with clindamycin antibiotics, and respectively selects suspected bacterial colonies for gram staining and biochemical identification. The method has long detection period and low accuracy of biochemical identification.
The method uses improved MRS selective culture medium to count lactobacillus, and selects 3 or more single colonies to perform smear microscopy and biochemical identification. The method has the advantages of long detection period, low selectivity and low biochemical identification accuracy.
Reference [ Molecular characterization of lactic acid bacteria in a transferred milk products using real-time quantitative PCR, International Journal of Food Microbiology,2004(97): 197-207.]The method utilizes specific primers to perform real-time fluorescence quantitative PCR (polymerase chain reaction) to detect the population level of lactobacillus casei (containing lactobacillus casei, lactobacillus paracasei and lactobacillus rhamnosus) in the fermented milk, and the detection limit is 1 multiplied by 103CFU/mL, primers were obtained by 16S rRNA gene design. The method can only identify the lactobacillus casei to the population levelLactobacillus casei) Lactobacillus paracasei (I)Lactobacillus paracasei) Lactobacillus rhamnosus (A), (B)Lactobacillus rhamnosus) And the like, only dead bacteria can be detected, and dead bacteria and live bacteria cannot be distinguished.
Identification and qualification ofLactobacillus casei strain Shirota in human feces with strain-specific primers derived from randomly amplified polymorphic DNA. International Journal of Food Microbiology, 2008(126): 210-215]The method utilizes specific primers to perform strain level detection on the Lactobacillus casei field strain in real-time fluorescent quantitative PCR, and the primers are obtained by RAPD test design. The method can only detect the field strains of the lactobacillus casei, can only detect dead bacteria, and cannot distinguish the dead bacteriaBacteria and live bacteria.
Literature [ Quantitative Real-Time PCR Analysis of FecalLactobacillusSpecies in Infants Receiving a Prebiotic Infant Formula. Applied and Environmental Microbiology, 2006,2359-2365]The method utilizes specific primers to carry out species level detection on the lactobacillus casei by real-time fluorescence quantitative PCR, and the primers are obtained by designing 16S-23S sections. The method can only detect pure strains, does not relate to products such as fermented milk and the like, can only detect dead bacteria, and cannot distinguish the dead bacteria from the live bacteria.
Literature [ biological bacteria survivor in chemical chemistry and modification of other lactic acid bacteria, Journal of Applied Microbiology, 2014 (116),1642-1656]In the method, PMA and specific primers are used for carrying out fluorescence quantitative PCR (polymerase chain reaction) to detect the population level of lactobacillus casei and lactobacillus paracasei in the cheese, and the primers are obtained by 16S rRNA gene design. The method can only identify the lactobacillus casei to the population levelLactobacillus casei) Lactobacillus paracasei (I)Lactobacillus paracasei) And the like.
By combining the prior art, the existing identification and detection of the lactobacillus casei of the compound lactobacillus fermented milk and the microbial inoculum product also has a space for improving the detection period, the accuracy of the detection result, the detection of the level of the species, the distinction of viable bacteria and dead bacteria, the interference of the product background and the like, so that how to design the identification and detection method of the lactobacillus casei of the compound lactobacillus fermented milk and the microbial inoculum product which considers the improvement of the above weak links becomes an important content to be solved by the invention.
The present invention has the following advantages.
High resolution, and no discrimination between the species Lactobacillus casei of the Lactobacillus casei group in conventional primer design: (Lacticaseibacillus casei) Lactobacillus paracasei (I)Lacticaseibacillus paracasei) Lactobacillus rhamnosus (II)Lacticaseibacillus rhamnosus) How to correctly test the cheese lactobacillus in the compound fermented milk and the microbial inoculum becomes a great challenge, and different from a 16S sequence with a shorter sequence, a whole genome sequence more completely shows the difference between bacteria, and simultaneouslyBy finding a core single copy gene, the range of the differential gene is effectively reduced, and based on the mode, a specific primer with higher resolution is successfully designed.
The method can provide the viable count of the target bacteria for rapid detection, further optimizes the strain detection means in the compound fermented milk and the microbial inoculum, and has the characteristics of rapidness and accuracy.
Disclosure of Invention
The invention adopts PMA-qPCR specific detection technology to establish the composite lactobacillus fermented milk and the cheese bacillus in the microbial inoculumLacticaseibacillus casei) A method for precisely identifying and quantitatively detecting species level viable bacteria in a targeted manner. Through optimizing the condition of PMA treatment on the compound lactobacillus fermented milk sample, PMA is combined with DNA in dead cells to reduce PCR signals to obtain genome DNA treated by PMA, specific amplification primers and probe sequences of a species to be detected are designed, qPCR amplification is utilized to detect whether a specific amplification curve is presented or not, and the problem of target active lactobacillus casei (Lactobacillus casei) in the compound lactobacillus fermented milk and microbial inoculum is solvedLacticaseibacillus casei) Specific detection and quantification. It is a first object of the invention to provide a method of using a single copyrsmEThe nucleotide sequence of the gene is shown as SEQ ID No.1, the specific PCR detection primers for identifying and detecting the lactobacillus casei in the lactobacillus composite fermented milk and the microbial inoculum are designed based on the specific molecular markers, the primers are a pair, the nucleotide sequence of the forward primer is shown as SEQ ID No.2, the nucleotide sequence of the reverse primer is shown as SEQ ID No.3, and the detailed information of the primers is shown in Table 1.
Table 1 cheesecaloides primer information.
The primer design and specificity verification comprise specific primer design, PCR amplification verification primer specificity and database comparison verification primer specificity.
1) And (3) designing a specific primer.
Collection of 12 strains of cheese bacilli casei from CICC database and NCBI Genome database (II)Lacticaseibacillus casei) A whole genome sequence, adopting Busco software to calculate the single copy gene set of each cheese lactobacillus to obtain 123 single copy candidate genes of the cheese lactobacillus, and further screening according to the specificity and base difference of the single copy genes in lactobacillus strains to obtain the target genersmEGene using Primer Premier 6.0 and Primer-Blast pairrsmEThe total number of gene design primers is 6, and 1 pair of primers is obtained by filtering and screening under the conditions of blast specificity, secondary structure, product length, primer temperature and the like and is used for PCR amplification verification.
2) PCR amplification verifies primer specificity.
Selecting 13 strains of representative fermented milk strains and model strains from different sources, different species and the same species, culturing to logarithmic growth phase after recovery, respectively extracting genome DNA of the 13 strains by using a bacterial genome extraction kit, and performing PCR amplification by using primer groups, namely, casei-F/casei-R and (Table 1) by using double distilled water as negative control. The total PCR reaction system is 25 μ L, including premix Taq 12.5 μ L, upstream and downstream primers (10 μmol/L) each 1.0 μ L, diluted DNA template 2 μ L, sterile ddH2O make up the volume to 25. mu.L. Amplification conditions: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 30 s, 35 cycles; and stretching for 10 min after 72 ℃. The results of PCR amplification of 13 strains showed that only Lactobacillus casei (B.casei) ((B.casei))Lacticaseibacillus casei) Specific amplification exists, other reference strains and negative control have no strip, and the result shows that,rsmEthe gene primer set has species-level specificity (FIG. 1).
3) And (4) comparing the database to verify the specificity of the primers.
The specificity of the primers in the bacterial Genome range was verified in RefSeq Genome database Blast (parameter: bacterio (taxi: 2)), and the primer set, casei-F/casei-R (Table 1), was only in Lactobacillus casei（Lacticaseibacillus casei) And Lactobacillus zeae: (Lacticaseibacillus zeae) The existence of the strain is that the corn cheese bacillus is not approved by the regulatory department of China for food, so the strain is used for foodrsmEThe gene primer set has species level specificity in strains for food, including Lactobacillus casei (Lactobacillus casei, and Lactobacillus casei)Lacticaseibacilluscaseigroup) paracaseobacterium casei (C.casei) ((R)Lacticaseibacillusparacasei) And Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus）。
The second purpose of the invention is to provide a method for detecting the specificity PCR detection primer of the cheese bacillus to carry out the PMA-qPCR detection of the composite lactobacillus fermented milk and the cheese bacillus in the microbial inoculum, which comprises the following steps.
1) Treatment with propidium azide bromide (PMA).
Diluting the thallus concentration to 10 with sterile physiological saline8CFU/mL. Adding PMA into 500 μ L bacterial solution to make the final concentration 50 μ M, dark incubating for 5min, mixing every 1 min, and exposing to LED light sensor (American Biotium PT-H18A) for 15 min.
2) And (3) inhibiting PCR amplification of dead bacteria.
Scraping anaerobic, solid cultured Lactobacillus casei at 37 deg.C for 48h ((R))Lacticaseibacillus casei) Adjusting the concentration of the bacteria to 10 with sterile normal saline8Bacterial suspension (OD) of CFU/mL620About 0.4), one part is live bacteria, and the other part is dead bacteria (water bath treatment at 80 ℃ for 20 min). PMA treatment was performed according to 1). Centrifuging at 12000 rpm for 10 min at room temperature, collecting thallus, and extracting genome DNA of live and dead bacteria. And (3) detecting the inhibition rate of PMA treatment conditions on the amplification of the DNA of the dead bacteria by qPCR.
Inhibition (%) = (1-C)Dead bacteria-PMA (+)/CDead bacteria-PMA (-))%
CDead bacteria-PMA (+): bacterial concentration (CFU/mL) of the sample to be detected treated by PMA; cDead bacteria-PMA (-): bacteria concentration (CFU/mL) of the test sample without PMA treatment.
3) And (3) extracting genome DNA.
Genomic DNA extraction was performed according to DNeasy Blood & Tissue Kit.
4) And (3) real-time fluorescent quantitative PCR detection.
The PCR amplification system is as follows: premix ExTaq 12.5. mu.L, upstream and downstream primers (10. mu. mol/L) each 1.0. mu.L, 50 XRox 0.5. mu.L, diluted DNA template 5. mu.L, sterile ddH2O make up the volume to 25. mu.L. The PCR amplification procedure was as follows: 95 ℃, 30, 1 cycle; fluorescence signal detection was performed at 95 ℃ for 10 s, 55 ℃ for 35 s, 72 ℃ for 24 s, 40 cycles.
5) And (6) drawing a standard curve.
Scraping anaerobic, solid cultured Lactobacillus casei at 37 deg.C for 48h ((R))Lacticaseibacillus casei) Adjusting the concentration of the bacteria to 10 with sterile normal saline8Bacterial suspension (OD) of CFU/mL620About 0.4), 500. mu.L of bacterial suspension was treated with PMA and then extracted with the method of 3). The extracted genomic DNA was diluted in 10-fold gradients and the corresponding Cq values were determined by qPCR to establish colony concentrations (log)10CFU/mL) and Cq value and is given by the formula E =10−1/s-1 calculating the amplification efficiency, wherein s is the slope of the standard curve. The standard curve is shown in FIG. 2, and the cheese bacillus primers meet the quantitative PCR requirements.
Table 2 cheesecaloides standard curve information.
6) And (6) judging the result.
Lactobacillus casei (L.casei) (II)Lacticaseibacillus casei) The primer casei-F/casei-R has specificity in species and self genome, the size of PCR amplification band of genome DNA is matched with that of the genome DNA of a model strain (positive control), and a qPCR dissolution curve is a single peak. Shows that the casei-F/casei-R can realize the cheese bacillus in the composite fermented milk ()Lacticaseibacillus casei) And (4) carrying out quantitative detection.
The third purpose of the invention is to provide the method for the composite lactobacillus fermentationCheese lactobacillus in fermented milk and microbial inoculum: (Lacticaseibacillus casei) Lactobacillus casei (A. casei)Lactobacillus casei) And (3) identifying the application of the detection.
Drawings
FIG. 1 is a primer-specific amplification-verification electrophoretogram, in which M-2K marker, N-blank control, 1-CICC 6163TLactobacillus delbrueckii subspecies bulgaricusLactobacillus delbrueckii subsp. bulgaricus2-CICC 10134R Lactobacillus delbrueckii subspecies bulgaricusLactobacillus delbrueckii subsp. bulgaricus3-CICC 10139R Lactobacillus bulgaricus subspeciesLactobacillus delbrueckii subsp. bulgaricus; 4-CICC 6222TStreptococcus thermophilusStreptococcus thermophilus5-CICC 10135R Streptococcus thermophilusStreptococcus thermophilus6-CICC 10138R Streptococcus thermophilusStreptococcus thermophilus7-CICC 10141R Streptococcus thermophilusStreptococcus thermophilus; 8-CICC 6263TLactobacillus paracaseiLacticaseibacillus paracasei; 9-CICC 6117TCheese bacillusLacticaseibacillus casei10-CICC 10136R cheese bacillusLacticaseibacillus casei; 11-CICC 6250TBifidobacterium animalis subspeciesBifidobacterium animalis subsp.animalis12-CICC 10137R bifidobacterium animalis subsp lactisBifidobacterium animalis subsp.lactis13-CICC 10140R bifidobacterium animalis subspecies lactisBifidobacterium animalis subsp. lactis。
FIG. 2 is a cheese bacilli qPCR standard curve (standard curve).
FIG. 3 is a cheese bacilli qPCR Amplification curve (Amplification curve).
FIG. 4 is a cheese bacilli qPCR Melting curve (Melding curve).
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1.
1) And detecting sample information.
Selling a certain brandFermented milk (production date: 202108170047, specification: 135 g/bottle, marked strain composition including Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus casei, marked lactobacillus viable count greater than or equal to 1 × 106 CFU/g）。
2) And detecting the specificity in the cheese bacillus genome by real-time fluorescent quantitative PCR.
Lactobacillus casei (a) is prepared from Lactobacillus caseiLacticaseibacillus casei) After CICC10136R is recovered, the strain is cultured to a logarithmic growth phase, bacterial genome DNA is respectively extracted by a bacterial genome extraction kit, double distilled water is used as negative control, and a primer group, namely, casei-F/casei-R (shown in table 1) is applied to carry out fluorescence quantitative PCR amplification. The total reaction system of the fluorescent quantitative PCR is 25 μ L, which comprises 12.5 μ L of premix ExTaq, 1.0 μ L of each of the upstream and downstream primers (10 μmol/L), 0.5 μ L of 50 XRox, 5 μ L of diluted DNA template, and sterile ddH2O to make up the volume to 25 μ L. Amplification conditions: 95 ℃, 30, 1 cycle; fluorescence signals were collected at 55 ℃ for 40 cycles of 95 ℃ 10 s, 55 ℃ 35 s, 72 ℃ 24 s, and melting curves were added at the end of the reaction. The specific real-time fluorescent quantitative PCR detection result of the primers in the genome shows that the melting curve of the product is single, which indicates thatrsmEThe gene primers were specific well within the genome (FIG. 2).
3) And (3) extracting genome DNA.
Genomic DNA extraction was performed according to DNeasy Blood & Tissue Kit.
4) And (5) drawing a sensitivity test and a standard curve.
The obtained genome DNA is diluted by 7 gradients in a 10-fold gradient manner, a Cq value corresponding to each dilution DNA is determined by qPCR, and a qPCR standard curve based on CFU is established by taking the logarithm value (log 10 CFU/mL) of the bacterial liquid concentration as an x axis and the Cq value as a y axis. According to the plate count results, Lactobacillus casei: (Lacticaseibacillus casei) The concentration of the culture was 3.7X 108CFU/mL, real-time fluorescent PCR detection of Lactobacillus casei: (Lacticaseibacillus casei) The dilution of (A) is in order from 3.7X 108CFU/mL~3.7×103CFU/mL, the results showed that the limit of detection (LOD) was 3,700CFU/mL, the logarithm of the bacterial liquid concentration was plotted on the abscissa, and the cycle count Cq was plotted on the ordinateThe standard curve (fig. 3) was constructed with a standard curve linear regression coefficient R2 of 1. Cheese bacillus y = -3.355x + 42.594; r2= 1; eff% = 98.63%.
5) Treatment of azide propidium bromide (PMA) and verification of inhibition rate of dead bacteria/live bacteria.
Scraping the cheese bacilli cultured in solid for 48h under anaerobic conditions at 37 ℃ ((C))Lacticaseibacillus casei) The cells were diluted to 108 CFU/mL with sterile physiological saline (OD 620: about 0.4), and divided into two portions, one portion was designated as viable cells, and the other portion was treated in a 80 ℃ water bath for 20min, and immediately cooled in an ice bath after being taken out, and designated as dead cells (no viable cell growth was detected by a plate colony counting method). Adding PMA solution into 500 μ L live and dead bacteria respectively to make the final concentration 50 μ M, dark incubating for 5min, mixing uniformly every 1 min, and exposing in LED photosensizer (American Biotium PT-H18A) for 15 min. The cells were collected by centrifugation at 12000 rpm for 15 min at room temperature.
Statistical software is used for carrying out one-way variance analysis on the difference of the Cq values before and after PMA treatment. Lactobacillus casei (L.casei) (II)Lacticaseibacillus casei) The final concentration of PMA was 50. mu. mol/L, and the dark incubation time was 15 min. The exposure time is 15 min, and the inhibition rate of PMA action conditions on the dead bacteria qPCR is as follows.
Table 3 inhibition rate of the PMA action conditions on the dead bacteria qPCR.
Note: PMA (-): no PMA effect is exerted; PMA (+): adding PMA to act.
Lactobacillus casei (L.casei) (II)Lacticaseibacillus casei) The final concentration of PMA is 50 mu mol/L, the dark incubation time is 15 min, and the exposure time is 15 min.
The effect of PMA conditions on live bacteria qPCR is as follows.
Table 4 effect of PMA action conditions on live bacteria qPCR.
Note: PMA (-): no PMA effect is exerted; PMA (+): adding PMA to act.
Non-heat-treated Lactobacillus casei (A), (B), (C), (Lacticaseibacillus casei) The concentration is 7.57X 107CFU/mL; after heat treatment at 80 ℃ for 20min, plates were counted for cheeseobacter casei (Lacticaseibacillus casei) The concentration was 0. The Cq values of qPCR of live bacteria without PMA treatment and live bacteria with PMA treatment are analyzed through t test, and the result shows that no significant difference exists between the two Cq values of the live bacteria (P)>0.05); the Cq values of the qPCR of the dead bacteria without PMA treatment and the qPCR of the dead bacteria with PMA treatment show that the two Cq values of the dead bacteria have significant difference (P)>0.05). The PMA-qPCR method can distinguish live bacteria from dead bacteria, so that the detection result of the PMA-qPCR method is more accurate (figure 4). The inhibition rate of PMA on dead bacteria/live bacteria meets the requirement of the expected application of the test.
6) And (3) real-time fluorescent quantitative PCR detection.
The PCR amplification system is as follows: premix ExTaq 12.5. mu.L, upstream and downstream primers (10. mu. mol/L) each 1.0. mu.L, 50 XRox 0.5. mu.L, diluted DNA template 5. mu.L, sterile ddH2O make up the volume to 25. mu.L. The PCR amplification procedure was as follows: 95 ℃, 30, 1 cycle; fluorescence signal detection was performed at 95 ℃ for 10 s, 55 ℃ for 35 s, 72 ℃ for 24 s, 40 cycles.
7) And (6) detecting the result.
Detecting the lactobacillus casei in the detected sample by qPCR (Lacticaseibacillus casei) Is substituted into the formula y = -3.355x +42.594, thereby obtaining the detection sample of the cheese bacillus (Cq = 25.70) (h = -3.355x + 42.594)Lacticaseibacillus casei) The number of viable bacteria is 1.09 × 106 CFU/mL。
8) Compared with the detection result of the traditional culture method.
Referring to the method of GB 4789.35-2016 food safety national standard food microbiology test lactobacillus test, MRS (vancomycin containing 50 ug/mL) selective culture medium is adopted to replace the culture medium in the standard, and the cheese lactobacillus (A) in the detected sample is subjected toLacticaseibacillus casei) The result of colony counting of (2.5X 10)6CFU/mL, PMA-qPCR results were in order of magnitude consistent with selective media plate colony counts.
Sequence listing
<110> research institute of food fermentation in China, Ltd
<120> method for rapidly identifying and detecting cheese bacillus in fermented milk
<141> 2021-09-16
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ggtgtcagca aaggcgataa agccgagtgg attgtccaaa aagcaaccga attaggcgct 300
gccaagatcg gcttttttaa tgcacagtgg ggcactgccc gctggccggc cgaacgggtt 360
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Claims (4)
1. By usingrsmEThe gene design of a specific PCR detection primer for identifying and detecting the lactobacillus casei in the compound lactobacillus fermented milk and the microbial inoculum is characterized in that,rsmEthe nucleotide sequence of the gene is shown as SEQ ID No.1, the specific PCR detection primers are a pair, the nucleotide sequence of the forward primer is shown as SEQ ID No.2, and the nucleotide sequence of the reverse primer is shown as SEQ ID No. 3.
2. The method for detecting the lactobacillus casei PMA-qPCR in the lactobacillus casei fermented milk and the microbial inoculum by using the specific PCR detection primer of the lactobacillus casei of claim 1, is characterized by comprising the following operation steps: 1) treating azide propidium bromide; 2) b, inhibiting dead bacteria PCR amplification; 3) extracting genome DNA; 4) drawing a standard curve; 5) and (3) real-time fluorescent quantitative PCR detection.
3. The method of claim 2, wherein in operation 1), the parameters of the azide propidium bromide treatment are as follows: adding 25mL of sterilized normal saline into 2.5g of fermented milk sample per sample to be detected, shaking and mixing uniformly to prepare a 1:10 sample detection solution, adding azido propidium bromide into the sample detection solution to react, adding the azido propidium bromide with the final concentration of 50 mu M, reacting for 5min at 25 ℃ in a dark place after mixing uniformly, taking out and exposing in an LED (light-emitting diode) photosensizer for 15 min, and collecting thalli after the light reaction is finished.
4. The method of claim 2, wherein in operation 5), real-time fluorescent quantitative PCR is used for detecting the cheese bacillusrsmEThe gene and PCR amplification system is as follows: premix ExTaq 12.5. mu.L, upstream and downstream primers 10. mu. mol/L each 1.0. mu.L, 50 XRox 0.5. mu.L, diluted DNA template 5. mu.L, sterile ddH2O make up the volume to 25 μ L; the PCR amplification procedure was as follows: 95 ℃, 30, 1 cycle; fluorescence signal detection was performed at 95 ℃ for 10 s, 55 ℃ for 35 s, 72 ℃ for 24 s, 40 cycles.
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