CN112301140B - Method for detecting staphylococcus aureus in microecological live bacteria product - Google Patents
Method for detecting staphylococcus aureus in microecological live bacteria product Download PDFInfo
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- ZDWVWKDAWBGPDN-UHFFFAOYSA-O propidium Chemical compound C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 ZDWVWKDAWBGPDN-UHFFFAOYSA-O 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a method for detecting staphylococcus aureus in a microecological live bacteria product, which comprises the steps of adding propidium azide and propidium bromide into a live bacteria product heavy suspension after a large amount of auxiliary materials are removed by pretreatment, fully mixing, carrying out dark incubation at 20-25 ℃ for 5-20min, and then placing under an LED lamp for exposure for 5-20min to obtain a pretreated live bacteria product treatment solution; extracting genome DNA of the pretreated living bacteria product as a template, performing qPCR reaction, measuring a Ct value, and when the Ct value is smaller than 31, measuring that the microecological living bacteria product contains staphylococcus aureus living bacteria; the invention provides a PMA-qPCR detection method for staphylococcus aureus in a live bacterial product for the first time, and the detection sensitivity is 10 4 CFU/ml can be detected within 4 hours, and the method has the advantages of high efficiency, rapidness, real time, accuracy and the like compared with methods such as pharmacopoeia, national standard and the like, and has good application prospect.
Description
Field of the art
The invention relates to a method for detecting staphylococcus aureus in a microecological live bacteria product.
(II) background art
The relationship between microbial flora and human health and diseases has become a current medical research hot spot, and the microecological live bacteria product has positive significance in the aspects of maintaining the microbial flora balance, enhancing the human immunity and the like, and has become one of important means for health care of people. Therefore, the quality of the live bacteria product is directly related to the safety and effectiveness of the product, and the quality of the live bacteria product is particularly important to carry out strict quality supervision and verification, quality control and evaluation and the like. Staphylococcus aureus is one of the major pathogens responsible for food-borne diseases, and food poisoning caused by it is becoming an increasingly common health problem worldwide. The method can be used for rapidly, accurately and effectively checking pathogenic bacteria staphylococcus aureus in the microecological live bacteria products, and is an important guarantee for quality verification and quality evaluation of the microecological live bacteria products.
The inspection methods adopted by the Chinese pharmacopoeia and the food safety national standard for the staphylococcus aureus in the microecological live bacteria products are all traditional culture methods. However, the culture method has the defects of long detection time, poor accuracy, low efficiency, large workload and the like, and can not detect the active pathogenic bacteria rapidly and accurately. Usually, the detection process of the traditional culture method needs 2-3 days, and the obtained detection data has certain hysteresis, so that the real-time monitoring and early warning of pathogenic bacteria can not be realized. Meanwhile, billions or even hundreds of billions of viable bacteria exist in the microecological viable bacteria product, the growth of staphylococcus aureus is often inhibited by high-concentration viable bacteria, the condition that the background viable bacteria cover the growth of target pathogenic bacteria after culture occurs, and great interference is generated to the detection of staphylococcus aureus.
The method of combining azide propidium bromide (propidium monoazide, PMA) with fluorescent quantitative PCR is mainly focused on detection of common food and clinical pathogenic bacteria at present, and detection of active pathogenic bacteria in microecological live bacteria products has not been reported. The microecological live bacteria product generally contains a large amount of auxiliary materials, and dead bacteria can be generated in the production process of the live bacteria product, such as tabletting, transportation or storage, and the combination of the azide propidium bromide nucleic acid dye and the like and DNA can be influenced.
(III) summary of the invention
The invention aims to provide a method for detecting staphylococcus aureus in a microecological live bacteria product, which combines a microporous membrane filtration method, azidopropionibacterium bromide and a fluorescent quantitative PCR technology, so that the method can eliminate the interference of a large amount of auxiliary materials in the live bacteria product on the detection method, can entrap microorganisms in a sample, can accurately and rapidly detect staphylococcus aureus live bacteria in the microecological live bacteria product, only needs 4 hours for screening and identifying the whole process, and solves the problem that the traditional method can not rapidly and accurately detect staphylococcus aureus in the microecological live bacteria product.
The technical scheme adopted by the invention is as follows:
the invention provides a method for detecting staphylococcus aureus in a microecological live bacteria product, which comprises the following steps:
(1) Sample pretreatment: adding living bacteria product into sterile physiological saline, homogenizing at 2000-10000rpm for 30s-60s (preferably 3500rpm for 30 s), filtering with polyether sulfone (PES) microporous membrane, collecting filtrate, centrifuging (preferably 8000rpm for 5 min), discarding supernatant, and re-suspending with sterile distilled water to obtain pretreated living bacteria product suspension;
(2) Pretreatment of azido propidium bromide: adding propidium azide and propidium bromide into the heavy suspension of the living bacteria product pretreated in the step (1), fully and uniformly mixing, carrying out dark incubation at 20-25 ℃ for 5-20min, and then placing the mixture in an LED lamp for exposure for 5-20min to obtain pretreated living bacteria product treatment liquid;
(3) Real-time fluorescent quantitative PCR detection:
extracting genome DNA of the living bacteria product pretreated in the step (2) as a template, performing qPCR reaction, measuring a Ct value, and when the Ct value is smaller than 31, measuring that the microecological living bacteria product contains staphylococcus aureus living bacteria;
an upstream primer: 5'-TTCTTCACGACTAAATAAACGCTC A-3';
a downstream primer: 5 'GGTACTACTAAAGATTATCAAGCGGCT-3';
probe sequence: 5'-ROX-CAGAACACAATGTTTCCGATGCAACGT-BHQ2-3'.
Further, in the step (1), the volume amount of the sterile physiological saline is 12-57-ml/3g, preferably 27ml/3g, based on the weight of the live bacteria preparation; the pore size of the polyether sulfone (PES) microporous filter membrane is 3.0-6.0 μm, preferably 5.0 μm.
Further, in step (2), the azido propidium bromide is added as a 400 μg/mL aqueous solution; the azido propidium bromide is added at a final concentration of 30-60. Mu.g/mL, preferably 40. Mu.g/mL.
Further, the incubation in the step (2) is preferably performed for 10min, and vortex mixing is performed every 5 min.
Further, the LED lamp in the step (2) emits light with the wavelength of 465-475nm, preferably is exposed for 15min, and is uniformly mixed every 5 min.
Further, in step (3), qPCR reaction system: 10. Mu.L of ProbeqPCR Mix (with UNG); the amount of each of the upstream and downstream primers was 0.4. Mu.L; probe 0.8 μl; 2. Mu.L of template DNA; ddH 2 O6.2. Mu.L, total volume 20. Mu.L.
Further, in step (3), qPCR reaction conditions: preheating at 25 ℃ for 10min; stage1 pre-denaturation: 95 ℃ for 30s; stage2PCR reaction: 95 ℃,5s,60 ℃,34s,40 cycles.
In the step (2) of the invention, the TaKaRa bacterial genome DNA extraction kit is adopted to extract the sample genome DNA. The microecological live bacteria product contains a large amount of auxiliary materials, and the live bacteria product can generate high-concentration dead bacteria in the production process (such as tabletting) and transportation or storage, thereby affecting the combination of the azide propidium bromide nucleic acid dye and the like and DNA. The interference of a large amount of auxiliary materials in the living bacteria product on the detection method can be eliminated by the microporous membrane pretreatment, and the microorganisms in the sample can be reserved. The method can inhibit the interference of dead bacteria DNA on the detection of staphylococcus aureus live bacteria by optimizing the concentration of the azide and propidium bromide and combining the azide and the propidium bromide with high-concentration dead bacteria in a sample.
Compared with the prior art, the invention has the beneficial effects that:
the invention provides a PMA-qPCR detection method for staphylococcus aureus in a live bacterial product for the first time, and the detection sensitivity is 10 4 CFU/ml can be detected within 4 hours, and the method has the advantages of high efficiency, rapidness, real time, accuracy and the like compared with methods such as pharmacopoeia, national standard and the like, and has good application prospect.
(IV) description of the drawings
FIG. 1 shows the effect of no treatment of live bacteria samples on the detection of dead bacteria of Staphylococcus aureus.
FIG. 2 shows the effect of PMA concentration on the detection of staphylococcus aureus dead bacteria.
FIG. 3 shows the effect of PMA concentration on the detection of Staphylococcus aureus.
FIG. 4 is a graph showing the effect of dark incubation time on the detection of staphylococcus aureus dead bacteria.
FIG. 5 is a graph showing the effect of PMA exposure time on the detection of staphylococcus aureus dead bacteria.
FIG. 6 shows the effect of PMA on the detection of viable bacteria of different concentrations of Staphylococcus aureus.
FIG. 7 is a graph showing the sensitivity of concentration detection of live Staphylococcus aureus.
FIG. 8 is a graph showing the specificity of detection of live staphylococcus aureus.
(fifth) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
example 1,
1. Preparation of viable bacteria suspension
Staphylococcus aureus (Staphylococcus aureus) CMCC (B) 26003 (purchased from the national food and drug verification institute) and staphylococcus epidermidis (Staphylococcus epidermidis) CMCC (B) 26069 (purchased from the national food and drug verification institute) were inoculated to TSB medium respectively, and cultured at 35 ℃ for 24 hours, to obtain a staphylococcus aureus viable bacterial suspension in the logarithmic growth phase and a staphylococcus epidermidis viable bacterial suspension respectively.
To determine the concentration of the cells in the viable cell suspension, 7 gradients were serially diluted by 10-fold in saline and the final dilution stage (1X 10 8 ) 1mL of a dish is evenly mixed with 20mL of TSA culture medium, and is cultured for 24 hours at 35 ℃ and then counted, wherein the concentration of the staphylococcus aureus viable bacteria suspension is 10 8 CFU/mL, the concentration of the staphylococcus epidermidis viable bacterial suspension is 10 8 CFU/mL。
TSB media formulation (g/L): tryptone 17.0, soyase 3.0, sodium chloride 5.0, dipotassium hydrogen phosphate 2.5, glucose 2.5, water as solvent and pH 7.3+/-0.2.
TSA media formulation (g/L): 15.0 parts of tryptone, 5.0 parts of soybean peptone, 5.0 parts of sodium chloride, 15.0 parts of agar and water as a solvent, wherein the pH value is 7.3+/-0.2.
EXAMPLE 2 preparation of dead bacterial suspension
1) Screening for thermal death time: 10mL of the staphylococcus aureus live bacterial suspension prepared in the method of the example 1 is respectively taken, and is immediately placed on ice for cooling after being treated for 0min, 10min, 20min, 30min, 40min and 50min by using a boiling water bath. The number of viable bacteria was determined by plate counting, and the minimum boiling water bath treatment time for aseptic growth on the plates was used as the optimal thermal lethal time.
10 8 After 40min of treatment of CFU/mL staphylococcus aureus live bacterial suspension by boiling water bath, the colony growth on the plate is counted. Treatment with boiling water bath for 40min was the optimal thermal death time.
2) Preparation of dead bacterial suspension: taking 10mL of staphylococcus aureus live bacterial suspension, boiling in water bath for 40min, centrifuging at 8000rpm for 5min, discarding supernatant, and re-suspending and precipitating with 10mL of sterile distilled water to obtain staphylococcus aureus dead bacterial suspension (10) 8 CFU/mL)。
EXAMPLE 3 influence of no treatment of live bacteria samples on PMA-qPCR determination of dead bacteria of Staphylococcus aureus
The experiment sample adopts bacillus licheniformis viable bacteria capsule (purchased from Zhejiang Beijing New pharmaceutical industry Co., ltd., viable bacteria concentration 10) 9 CFU/g, 90% of auxiliary materials). 3.0g of bacillus licheniformis viable bacteria capsule content is aseptically weighed and added into 27.0ml of aseptic normal saline diluent, homogenized for 30s at 3500rpm, and prepared into 10% of test solution with the mass concentration of 10% 9 CFU/10mL。
The staphylococcus aureus dead bacteria suspension (10) prepared by the method of example 2 8 CFU/mL) were placed in 2 sets of centrifuge tubes, respectively, and test tube 1 was filled with no test solution, and 1.5mL of sterile distilled water was added to a constant volume of 2mL; the test tube 2 was added with 10mL of the test solution prepared in the above manner, mixed well, centrifuged at 8000rpm for 5min, the supernatant was discarded, and the precipitate was resuspended to a constant volume of 2mL with sterile distilled water. Adding 400 mug/mL of PMA aqueous solution into each centrifuge tube, enabling the final concentration of PMA to be 100 mug/mL, carrying out dark incubation at 20-25 ℃ for 15min after uniformly mixing, carrying out vortex mixing at intervals of 5min, placing the centrifuge tubes into a photolyzer (LED lamp with emission wavelength of 65-475 nm) for exposure for 20min, and carrying out uniform mixing at intervals of 5 min. DNA was extracted using the TaKaRa bacterial genomic DNA extraction kit. The concentration of the extracted DNA is measured by a Biodrop ultramicro protein nucleic acid analyzer, the concentration of the extracted DNA in the experimental group 1 is 851ng/mL, and the concentration of the extracted DNA in the experimental group 2 is 14ng/mL, so that a large amount of auxiliary materials contained in the test solution can influence the extraction amount of the DNA. qPCR detection was then performed to obtain Ct values.
PMA-qPCR reaction system (20. Mu.L): 10. Mu.L of Probe qPCR Mix (with UNG); the upstream and downstream primers were each 0.4. Mu.mL is; probe 0.8 μl; 2. Mu.L of template DNA; ddH 2 O 6.2μL。
An upstream primer: 5'-TTCTTCACGACTAAATAAACG CTCA-3';
a downstream primer: 5'GGTACTACTAAAGAT TATCAAGACGGCT-3';
probe sequence: 5'-ROX-CAGAACA CAATGTTTCCGATGCAACGT-BHQ2-3'.
PMA-qPCR reaction conditions: preheating at 25 ℃ for 10min; stage1 pre-denaturation: 95 ℃ for 30s; stage2PCR reaction: fluorescence signals were collected simultaneously for 34s,40 cycles at 95 ℃,5s,60 ℃.
As can be seen from FIG. 1, the Ct value of experimental group 1 was 31.254, which indicates that PMA almost completely inhibited Staphylococcus aureus dead bacteria and was not subjected to fluorescent quantitative PCR amplification. The Ct value of experiment group 2 is 21.208, which shows that auxiliary materials in the bacillus licheniformis viable bacteria preparation sample interfere with the combination of PMA and dead bacteria DNA, and influence the combination effect, so that the Ct value is reduced. Therefore, the auxiliary materials in the sample need to be removed, and the influence of the auxiliary materials on DNA extraction and the influence of the auxiliary materials on the combination of PMA and dead bacteria DNA are eliminated.
EXAMPLE 4 pretreatment of live bacteria sample
10ml of each of the test solutions having a mass concentration of 10% prepared in example 3 was collected, and each of the test solutions was filtered using a Mixed Cellulose Ester (MCE) microporous filter membrane (diameter: 50 mm) having a pore diameter of 0.45. Mu.m, a polyether sulfone (PES) microporous filter membrane (diameter: 50 mm) having a pore diameter of 1.2 μm and 5.0. Mu.m, and a nylon microporous filter membrane (diameter: 50 mm) having a pore diameter of 3.0 μm, 5.0 μm and 8.0. Mu.m, respectively, using an FC502 sterile filter (available from Thai Biotechnology Co., ltd.) and the auxiliary materials were trapped on the membrane. And respectively taking the filtrate to count viable bacteria, wherein the viable bacteria count of the filtrate filtered by the mixed cellulose ester microporous filter membrane with the thickness of 0.45 mu m is 0CFU/g, and all the viable bacteria and auxiliary materials are trapped by the membrane. Wherein the three filtrates filtered by 5.0 μm microporous membrane and 8.0 μm microporous membrane have highest viable count of 10 9 CFU/g, and compared with a weighing method, the 5.0 mu m polyether sulfone microporous filter membrane has the highest auxiliary material filtering rate. The test results and the characteristics of membranes made of different materials are combined, and a 5.0 mu m polyether sulfone microporous filter membrane (with the diameter of 50 mm) is selected for the test.
Collecting 5.0 μm filtrate from microporous membrane filtration with 8000rpm, centrifuging for 5min, discarding supernatant, and re-suspending with sterile distilled water to 500 μl to obtain Bacillus licheniformis viable bacteria capsule sample suspension (10) 9 CFU/mL), i.e., the pretreated product.
Example 5 optimization of the concentration of propidium azide (PMA)
The staphylococcus aureus live bacterial suspensions (10) prepared in the method of example 1 were taken respectively 8 CFU/mL) and Staphylococcus aureus dead bacterial suspension (10) prepared by the method of example 2 8 500 mu L of each CFU/mL were placed in different centrifuge tubes, and 500 mu L of the Bacillus licheniformis viable cell capsule sample suspension (10) prepared by the method of example 4 was added respectively 9 CFU/mL), centrifuging at 8000rpm for 5min, discarding the supernatant, and re-suspending the pellet with sterile distilled water to a volume of 0.5mL. Adding 400 mug/mL of PMA aqueous solution into each centrifuge tube respectively to make final concentrations of PMA 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 and 110 mug/mL, uniformly mixing, then dark incubating at 20-25 ℃ for 15min, uniformly mixing every 5min by vortex, placing the centrifuge tube in a photolyzer (LED lamp with emission wavelength of 65-475 nm) for exposure for 20min, and uniformly mixing every 5 min. DNA was extracted using the TaKaRa bacterial genomic DNA extraction kit. The concentration of the extracted DNA is measured by a Biodrop ultramicro protein nucleic acid analyzer, and the result is 100-1000 ng/mL. qPCR detection was then performed to obtain Ct values. The qPCR reaction system, primer probe and reaction conditions were the same as in example 3.
As can be seen from fig. 2, the inhibition rate against dead bacteria gradually increased with increasing PMA concentration. When the PMA concentration reached 40. Mu.g/mL, ct was 31.416 and after 40. Mu.g/mL, ct was not substantially changed (P >0.05, no significant difference), at which time the PMA inhibition was about 99% (average Ct of 8 points from 40. Mu.g/mL to 110. Mu.g/mL was 31.770, inhibition was 31.416/31.770 =98.9%).
As is clear from FIG. 3, the Ct value of the living bacteria amplified initially was unchanged with the increase of the PMA concentration, and the Ct value gradually increased when the PMA concentration exceeded 60. Mu.g/mL, indicating that PMA was able to significantly inhibit qPCR amplification of living bacteria at this concentration, and the higher the concentration, the more significant the inhibition.
Therefore, when the concentration of PMA is 40 mug/mL, the combination of PMA and dead bacillus licheniformis in the sample can be deducted, the amplification of the DNA of the dead bacillus aureus can be effectively inhibited, and the PCR amplification of the DNA of the live bacillus aureus can not be influenced. 40. Mu.g/mL was chosen as the optimal working concentration for PMA.
Example 6 optimization of propidium azide-bromide dark incubation time
The staphylococcus aureus dead bacteria suspensions (10) prepared in the method of example 2 were taken respectively 8 CFU/mL) 500. Mu.L was placed in 1.5mL centrifuge tubes, and each centrifuge tube was charged with 500. Mu.L of the Bacillus licheniformis viable bacteria capsule sample suspension (10) prepared by the method of example 4 9 CFU/mL), 8000rpm, discarding the supernatant, re-suspending the pellet with sterile distilled water to a volume of 0.5mL. Adding 400 mug/mL PMA aqueous solution into each centrifuge tube to make the final concentration of the PMA aqueous solution 40 mug/mL, carrying out vortex mixing uniformly, setting dark incubation time to be 0, 5, 10, 15, 20, 25 and 30min respectively, carrying out dark incubation at 20-25 ℃, carrying out vortex mixing uniformly every 5min, placing the centrifuge tube on a photolyzer for exposure for 20min, and carrying out uniform mixing every 5 min. Genomic DNA was then extracted and subjected to qPCR analysis by the method of example 5. The qPCR reaction system, primer probe and reaction conditions were the same as in example 3.
As can be seen from fig. 4, the Ct value does not significantly change with the increase of the dark incubation time, and 10min was selected as the dark incubation time in consideration of the durability of the method.
EXAMPLE 7 optimization of propidium azide Exposure time
mu.L of the dead staphylococcus aureus suspension (10) prepared by the method of example 2 was taken respectively 8 CFU/mL) were placed in 1.5mL centrifuge tubes, and 500. Mu.L of the Bacillus licheniformis viable cell sample suspension (10) prepared by the method of example 4 was added to each centrifuge tube 9 CFU/mL), 8000rpm, discarding the supernatant, re-suspending the pellet with sterile distilled water to a volume of 0.5mL. Adding 400 mug/mL PMA aqueous solution into each centrifuge tube to make the final concentration of the PMA aqueous solution 40 mug/mL, carrying out vortex mixing uniformly, then respectively placing the mixture into a dark incubation time of 20-25 ℃ for 10min, carrying out vortex mixing uniformly every 5min, and respectively exposing the mixture to light for 0, 5, 10, 15, 20, 25 and 30min on a photolyzer, and carrying out vortex mixing uniformly every 5 min. Genomic DNA was extracted by the method of example 4 and then subjected to qPCR analysis. qPCR reaction system, primer probe and reaction condition are the same as those of the real sampleExample 3.
As can be seen from fig. 5, the Ct value gradually increases with the increase of the exposure time, and the plateau phase is entered after the exposure time increases to 5 min. Considering the durability of the method and the fact that a certain amount of dead bacteria is contained in a bacillus licheniformis viable bacteria capsule sample, effective combination of PMA and the dead bacteria is ensured, and 10min is selected as exposure time.
EXAMPLE 8 validation of different concentrations of Staphylococcus aureus dead bacteria
The staphylococcus aureus dead bacterial suspension (10) prepared by the method of example 2 8 CFU/mL) was added to the live staphylococcus aureus suspension prepared in example 1, and the live staphylococcus aureus suspension was prepared in a ratio of 0%, 1%, 10%, 25%, 50% and 100%, 500. Mu.L each was placed in a 1.5mL centrifuge tube, and 500. Mu.L of the live bacillus licheniformis capsule sample suspension prepared in example 4 was added to each centrifuge tube (10) 9 CFU/mL), 8000rpm, discarding the supernatant, re-suspending the pellet with sterile distilled water to a volume of 0.5mL. Adding 400 mug/mL PMA aqueous solution into each centrifuge tube to make the final concentration 40 mug/mL, mixing uniformly by vortex, then placing the mixture into a dark incubation time of 20-25 ℃ for 10min respectively, mixing uniformly by vortex every 5min, exposing the mixture on a photolyzer for 10min respectively, and mixing uniformly by vortex every 5 min. Genomic DNA was extracted and qPCR detected using the method of example 4. The qPCR reaction system, primer probe and reaction conditions were the same as in example 3.
Under the same conditions, no PMA was added as a control.
As can be seen from FIG. 6, in the experimental group without PMA, the Ct value was kept constant with the increase of the specific row of viable bacteria. In the experimental group with PMA, the Ct value decreases with the increase of the specific list of living bacteria and is higher than that of the sample which is not treated by PMA. The Ct values of the samples not treated with PMA and the samples treated with PMA were not different when the viable bacteria ratio was 100%. The PMA can be effectively combined with dead bacteria DNA under the experimental conditions established by the method, so that the subsequent DNA amplification is inhibited, the false positive result is avoided, meanwhile, the detection of living bacteria is not influenced, and the false negative result is avoided.
Example 9 sensitivity study
10 prepared in example 1 Using physiological saline 8 CFU/mLSequentially performing 10-time gradient dilution on the staphylococcus aureus viable bacteria suspension to obtain a concentration of 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 、10 0 CFU/mL bacterial liquid is respectively taken 500 mu L in 1.5mL centrifuge tubes, and 0.5mL of bacillus licheniformis viable bacteria capsule sample suspension (10) prepared by the method of example 4 is respectively added into each centrifuge tube 9 CFU/mL), 8000rpm, discarding the supernatant, and re-suspending the pellet with sterile distilled water to a volume of 0.5mL, respectively. Adding 400 mug/mL PMA aqueous solution into each centrifuge tube to make the final concentration 40 mug/mL, mixing uniformly by vortex, then placing the mixture into a dark incubation time of 20-25 ℃ for 10min respectively, mixing uniformly by vortex every 5min, exposing the mixture on a photolyzer for 10min respectively, and mixing uniformly by vortex every 5 min. Genomic DNA was extracted and qPCR detected using the method of example 4. The qPCR reaction system, primer probe and reaction conditions were the same as in example 3.
As can be seen in FIG. 7, the concentration of the viable staphylococcus aureus suspension at 10 4 ～10 8 Exhibits good linear relationship with Ct value in CFU/mL range (R 2 0.9916), the standard curve is y= -2.8905x+34.025, and the detection limit is 10 4 CFU/mL。
Example 10 specificity Studies
1mL of each of the staphylococcus aureus live bacterial suspension and the staphylococcus epidermidis bacterial suspension prepared by the method of example 1 is taken and respectively placed in 2mL centrifuge tubes, and 500 mu L of the bacillus licheniformis live bacterial capsule sample suspension (10) prepared by the method of example 4 is respectively added into each centrifuge tube 9 CFU/mL), 8000rpm, discarding the supernatant, re-suspending the pellet with sterile distilled water to a volume of 0.5mL. Adding 400 mug/mL PMA aqueous solution into each centrifuge tube to make the final concentration of the PMA aqueous solution 40 mug/mL, mixing the mixture uniformly by vortex, then placing the mixture into a dark incubation time of 20-25 ℃ for 10min respectively, mixing the mixture uniformly by vortex every 5min, exposing the mixture on a photolyzer for 10min respectively, and mixing the mixture uniformly by vortex every 5 min. Genomic DNA was extracted by the method of example 4 and then subjected to qPCR analysis. The qPCR reaction system, primer probe and reaction conditions were the same as in example 3.
The fluorescent amplification signal of staphylococcus aureus has obvious exponential phase, the Ct value is 16.312, and as can be seen from figure 8, the staphylococcus epidermidis has no obvious fluorescent amplification curve, and the detection specificity of the method on staphylococcus aureus is high.
Sequence listing
<110> Zhejiang province food and drug inspection institute
<120> method for detecting staphylococcus aureus in microecological live bacteria product
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 25
<212> DNA
<213> Unknown (Unknown)
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ttcttcacga ctaaataaac gctca 25
<210> 2
<211> 28
<212> DNA
<213> Unknown (Unknown)
<400> 2
ggtactacta aagattatca agacggct 28
<210> 3
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<212> DNA
<213> Unknown (Unknown)
<400> 3
cagaacacaa tgtttccgat gcaacgt 27
Claims (4)
1. A method for detecting staphylococcus aureus in a microecological live bacteria product is characterized by comprising the following steps:
(1) Sample pretreatment: adding the live bacteria product into sterile normal saline, homogenizing, filtering with polyether sulfone microporous membrane, collecting filtrate, centrifuging, discarding supernatant, and re-suspending and precipitating with sterile distilled water to obtain pretreated live bacteria product heavy suspension; the aperture of the polyethersulfone microporous filter membrane is 5.0 mu m; the live bacteria product is bacillus licheniformis live bacteria capsule, and the live bacteria concentration is 10 9 CFU/g, and the auxiliary material accounts for 90 percent;
(2) Pretreatment of azido propidium bromide: adding propidium azide and propidium bromide into the heavy suspension of the living bacteria product pretreated in the step (1), fully and uniformly mixing, performing dark incubation at 20-25 ℃ for 10min, and then placing the mixture under an LED lamp for exposure for 10min to obtain pretreated living bacteria product treatment liquid; the azido propidium bromide is added in the form of 400 mug/mL aqueous solution; the final concentration of the azido propidium bromide added is 40 mug/mL;
(3) Real-time fluorescent quantitative PCR detection:
extracting genome DNA of the living bacteria product pretreated in the step (2) as a template, performing qPCR reaction, measuring a Ct value, and when the Ct value is smaller than 31, measuring the living bacteria of staphylococcus aureus in the microecological living bacteria product to be measured;
an upstream primer: 5'-TTCTTCACGACTAAATAAACGCTC A-3';
a downstream primer: 5'-GGTACTACTAAAGATTATCAAGACGGCT-3';
probe sequence: 5'-ROX-CAGAACACAATGTTTCCGATGCAACGT-BHQ2-3';
qPCR reaction conditions: preheating at 25 ℃ for 10min; stage1 pre-denaturation: 95 ℃ for 30s; stage2PCR reaction: 95 ℃,5s,60 ℃,34s,40 cycles.
2. The method of claim 1, wherein in step (1), the sterile physiological saline is used in an amount of 12 to 57ml/3g based on the weight of the live bacteria preparation.
3. The method of claim 1, wherein in step (2), the LED lamps emit wavelengths 465-475 nm.
4. The method of claim 1, wherein in step (2), the LED lamp is exposed for 15min, and mixed uniformly every 5 min.
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