CN112301140A - 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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- C—CHEMISTRY; METALLURGY
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- 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
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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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a method for detecting staphylococcus aureus in a microecological live bacterial product, which comprises the steps of adding azide propidium bromide into a live bacterial product heavy suspension liquid after a large amount of auxiliary materials are removed in a pretreatment way, fully and uniformly 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 bacterial product treatment liquid; extracting the pretreated live bacteria product genome DNA as a template, carrying out qPCR reaction to obtain a Ct value, and when the Ct value is less than 31, obtaining that the microecological live bacteria product to be detected contains staphylococcus aureus live bacteria; the invention provides a PMA-qPCR detection method of staphylococcus aureus in viable bacteria products for the first time, and the detection sensitivity is 104CFU/ml, within 4hThe method can complete detection, has the advantages of high efficiency, rapidness, real time, accuracy and the like, and has good application prospect compared with methods such as pharmacopoeia, national standard and the like.
Description
(I) technical field
The invention relates to a method for detecting staphylococcus aureus in a microecological live bacterium product.
(II) background of the invention
The relationship between microbial flora and human health and diseases has become a hotspot of current medical research, and the microbial live bacteria product has positive significance in maintaining flora balance, enhancing human immunity and the like, and becomes one of important means for health care of people. Therefore, the quality of the live bacterial product is directly related to the safety and effectiveness of the product, and strict quality supervision, verification, quality control, evaluation and the like are particularly important. Staphylococcus aureus is one of the leading pathogens causing food-borne diseases, and its food poisoning is becoming a worldwide public health problem. Can quickly, accurately and effectively check pathogenic bacteria staphylococcus aureus in the micro-ecological viable bacteria product, and is an important guarantee for quality verification and quality evaluation of the micro-ecological viable bacteria product.
The inspection methods adopted by the Chinese pharmacopoeia and the national food safety standard for staphylococcus aureus in microecological live bacteria products are both traditional culture methods. However, the culture method has the defects of long detection time, poor accuracy, low efficiency, large workload and the like, so that the active pathogenic bacteria cannot be quickly and accurately detected. Generally, the detection process of the traditional culture method needs 2-3 days, the obtained detection data have certain hysteresis, and real-time monitoring and early warning on pathogenic bacteria cannot be realized. Meanwhile, the micro-ecological viable bacteria product contains billions or even billions of viable bacteria, the high-concentration viable bacteria often inhibit the growth of staphylococcus aureus, the situation that the growth of target pathogenic bacteria is covered by background viable bacteria after culture occurs, and great interference is generated on the detection of the staphylococcus aureus.
The method of combining propidium azide bromide (PMA) with fluorescence quantitative PCR is used for live bacteria detection, which mainly focuses on detection of common food and clinical pathogenic bacteria at present, and detection of active pathogenic bacteria in a microecological live bacteria product is not reported yet. 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.
Disclosure 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, azide propidium bromide and a fluorescent quantitative PCR technology, can eliminate the interference of a large amount of auxiliary materials in the live bacteria product on the detection method, can retain microorganisms in a sample, can accurately and quickly detect the staphylococcus aureus live bacteria in the microecological live bacteria product, only needs 4 hours for screening and identification in the whole process, and solves the problem that the traditional method can not quickly and accurately detect the 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 bacterium product, which comprises the following steps:
(1) sample pretreatment: adding the viable bacteria product into sterile physiological saline, homogenizing at 2000-10000rpm for 30s-60s (preferably 3500rpm for 30s), filtering with a Polyethersulfone (PES) microporous filter membrane, collecting the filtrate, centrifuging (preferably 8000rpm for 5min), discarding the supernatant, and resuspending the precipitate with sterile distilled water to obtain a pretreated viable bacteria product resuspension solution;
(2) pre-treating azide propidium bromide: adding azide propidium bromide into the live bacteria product heavy suspension pretreated in the step (1), fully and uniformly mixing, carrying out dark incubation at 20-25 ℃ for 5-20min, and then placing in an LED lamp for exposure for 5-20min to obtain pretreated live bacteria product treatment liquid;
(3) real-time fluorescent quantitative PCR detection:
extracting the genome DNA of the viable bacteria product pretreated in the step (2) as a template, carrying out qPCR reaction to obtain a Ct value, and when the Ct value is less than 31, determining that the microbial viable bacteria product to be detected contains viable staphylococcus aureus;
an upstream primer: 5'-TTCTTCACGACTAAATAAACGCTC A-3', respectively;
a downstream primer: 5 'GGTACTACTAAAGATTATCAAGACGGCT-3';
the probe sequence is as follows: 5 '-ROX-CAGAACACAATGTTTCCGATGCAACGT-BHQ 2-3'.
Further, in the step (1), the volume dosage of the sterile physiological saline is 12-57-ml/3g, preferably 27ml/3g based on the weight of the viable bacteria product; the pore diameter of the Polyethersulfone (PES) microporous filter membrane is 3.0-6.0 μm, and preferably 5.0 μm.
Further, in the step (2), the azido propidium bromide is added in the form of 400 mu g/mL aqueous solution; the final concentration of the azido propidium bromide is added to be 30-60 mug/mL, and preferably 40 mug/mL.
Further, preferably, the incubation in step (2) is performed for 10min, and vortexed and mixed every 5 min.
Further, the LED lamp in the step (2) emits light with a wavelength of 465-475nm, preferably is exposed for 15min, and is uniformly mixed every 5 min.
Further, in step (3), the qPCR reaction system: ProbeqPCR Mix (with UNG) 10. mu.L; the upstream primer and the downstream primer are respectively 0.4 mu L; probe 0.8 μ L; 2 μ L of template DNA; ddH2O6.2. mu.L, total volume 20. mu.L.
Further, in step (3), the qPCR reaction conditions: preheating at 25 deg.C for 10 min; stage1 pre-denaturation: at 95 ℃ for 30 s; stage2PCR reaction: 95 ℃,5s, 60 ℃,34s, 40 cycles.
In the step (2), the TaKaRa bacterial genome DNA extraction kit is adopted to extract the sample genome DNA. The microecological viable bacteria product contains a large amount of auxiliary materials, and the viable bacteria product can generate high-concentration dead bacteria in the production process (such as tabletting) and transportation or storage, so that the combination of azide propidium bromide nucleic acid dye and the like and DNA is influenced. Through the filtration pretreatment of the microporous membrane, the interference of a large amount of auxiliary materials in the live bacteria product on the detection method can be eliminated, and the microorganisms in the sample can be retained. The concentration of the azide propidium bromide is optimized to be combined with high-concentration dead bacteria in a sample, so that the interference of dead bacteria DNA on the detection of the viable bacteria of staphylococcus aureus can be inhibited.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a PMA-qPCR detection method of staphylococcus aureus in viable bacteria products for the first time, and the detection sensitivity is 104CFU/ml can be detected within 4h, and compared with methods such as pharmacopoeia and national standard, the method has the advantages of high efficiency, rapidness, real time, accuracy and the like, and has good application prospectAnd (5) landscape.
(IV) description of the drawings
FIG. 1 shows the effect of no treatment of viable bacteria samples on the detection of dead bacteria in Staphylococcus aureus.
FIG. 2 shows the effect of PMA concentration on the detection of Staphylococcus aureus death.
FIG. 3 is a graph showing the effect of PMA concentration on viable Staphylococcus aureus detection.
FIG. 4 shows the effect of dark incubation time on Staphylococcus aureus necropsy detection.
FIG. 5 shows the effect of PMA exposure time on Staphylococcus aureus sterility detection.
FIG. 6 shows the effect of PMA on viable Staphylococcus aureus detection at different concentrations.
FIG. 7 is a graph showing the sensitivity of viable bacteria concentration detection in Staphylococcus aureus.
FIG. 8 is a graph showing the specificity of viable bacteria detection in Staphylococcus aureus.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
examples 1,
1. Preparation of viable bacterial suspension
Respectively inoculating Staphylococcus aureus (Staphylococcus aureus) CMCC (B)26003 (purchased from China food and drug testing institute) and Staphylococcus epidermidis (Staphylococcus epidermidis) CMCC (B)26069 (purchased from China food and drug testing institute) to a TSB culture medium, and culturing at 35 ℃ for 24h to respectively obtain a Staphylococcus aureus viable bacteria suspension and a Staphylococcus epidermidis viable bacteria suspension in a logarithmic growth phase.
To determine the concentration of the viable bacteria in the viable bacteria suspension, the suspension was sequentially diluted with physiological saline 10 times in order to obtain 7 gradients, and the final dilution stage (1X 10)8)1mL of the suspension is injected into a dish, mixed with 20mL of TSA culture medium, cultured at 35 ℃ for 24h and counted, wherein the concentration of the staphylococcus aureus viable bacteria suspension is 108CFU/mL, the viable bacteria suspension concentration of staphylococcus epidermidis is 108CFU/mL。
TSB medium formulation (g/L): tryptone 17.0, soybean peptone 3.0, sodium chloride 5.0, dipotassium hydrogen phosphate 2.5, glucose 2.5 and water as solvent, wherein the pH value is 7.3 +/-0.2.
TSA medium formulation (g/L): tryptone 15.0, soybean peptone 5.0, sodium chloride 5.0, agar 15.0, solvent water, pH 7.3 + -0.2.
Example 2 preparation of a suspension of dead bacteria
1) Screening of thermally induced dead time: 10mL of the live staphylococcus aureus suspension prepared in the example 1 is taken respectively, treated by boiling water bath for 0, 10, 20, 30, 40 and 50min, and then immediately placed on ice for cooling. The viable count was determined by plate counting method, and the minimum boiling water bath treatment time for aseptic growth on the plate was taken as the optimal thermal death time.
108And (3) carrying out boiling water bath treatment on the CFU/mL staphylococcus aureus viable bacteria suspension for 40min, and counting the growth of aseptic colonies on the flat plate. The optimal thermal dead time is 40min after boiling water bath treatment.
2) Preparation of a dead bacteria suspension: treating 10mL of viable Staphylococcus aureus suspension with boiling water bath for 40min, centrifuging at 8000rpm for 5min, discarding supernatant, and resuspending the precipitate with 10mL of sterile distilled water to obtain dead Staphylococcus aureus suspension (10%8CFU/mL)。
Example 3 Effect of non-treatment of viable bacteria samples on PMA-qPCR assay of dead bacteria of Staphylococcus aureus
The experimental sample adopts a bacillus licheniformis viable bacteria capsule (purchased from new pharmaceutical industries, Ltd., Jing, Zhejiang, with viable bacteria concentration of 10)9CFU/g, 90% of auxiliary materials). Aseptically weighing 3.0g of content of viable Bacillus licheniformis capsule, adding into 27.0ml of aseptic normal saline diluent, homogenizing at 3500rpm for 30s, and making into 10% test solution, 109CFU/10mL。
A dead Staphylococcus aureus suspension (10) prepared in example 2 was used8CFU/mL) are respectively placed in 2 groups of centrifuge tubes, no test solution is added into the experimental tube 1, and 1.5mL of sterile distilled water is added to the experimental tube to reach 2mL of constant volume; the experiment tube 2 is added with 10mL of test solution prepared by the method and evenly mixed, centrifuged for 5min at 8000rpm, the supernatant is discarded, and the sediment is resuspended by sterile distilled water to reach 2 mL. Adding 400 mu g/mL of PMA aqueous solution into each centrifuge tube to ensure that PM is addedAnd A is 100 mug/mL, after being mixed uniformly, the mixture is incubated in dark at 20-25 ℃ for 15min, the mixture is vortexed and mixed uniformly every 5min, the centrifuge tube is placed in a photolysis instrument (an LED lamp with 65-475nm emission wavelength) to be exposed for 20min, and the mixture is mixed uniformly every 5 min. DNA is extracted by adopting a TaKaRa bacterial genome 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 of the experimental group 1 is 851ng/mL, and the concentration of the extracted DNA of the experimental group 2 is 14ng/mL, so that the extraction amount of the DNA can be influenced by a large amount of auxiliary materials contained in the test solution. Then qPCR detection is carried out to obtain Ct value.
PMA-qPCR reaction (20. mu.L): probe qPCR Mix (with UNG) 10. mu.L; the upstream primer and the downstream primer are respectively 0.4 mu L; probe 0.8 μ L; 2 mu L of template DNA; ddH2O 6.2μL。
An upstream primer: 5'-TTCTTCACGACTAAATAAACG CTCA-3', respectively;
a downstream primer: 5 'GGTACTACTAAAGAT TATCAAGACGGCT-3';
the probe sequence is as follows: 5 '-ROX-CAGAACA CAATGTTTCCGATGCAACGT-BHQ 2-3'.
PMA-qPCR reaction conditions: preheating at 25 deg.C for 10 min; stage1 pre-denaturation: at 95 ℃ for 30 s; stage2PCR reaction: fluorescence signals were collected simultaneously for 40 cycles at 95 ℃,5s, 60 ℃,34 s.
As is clear from FIG. 1, the Ct value of test group 1 was 31.254, indicating that PMA almost completely inhibited Staphylococcus aureus and that no fluorescent quantitative PCR amplification was performed. The Ct value of the experimental group 2 is 21.208, which indicates that the auxiliary material in the live bacillus licheniformis preparation sample interferes with the combination of PMA and dead bacteria DNA, influences the combination effect and causes the Ct value to be reduced. Therefore, it is necessary to remove the auxiliary materials from the sample to eliminate the influence of the auxiliary materials on the DNA extraction and the combination of PMA and the DNA of dead bacteria.
Example 4 live bacteria sample pretreatment
10ml of each of 6 parts of 10% by mass test solutions prepared in example 3 were each prepared using a Mixed Cellulose Ester (MCE) microporous filter membrane (diameter 50mm) having a pore size of 0.45. mu.m, a polyether sulfone (PES) microporous filter membrane (diameter 50mm) having a pore size of 1.2 μm and 5.0. mu.m, and a nylon microporous filter membrane (diameter 50mm) having a pore size of 3.0. mu.m, 5.0. mu.m and 8.0. mu.m, respectively, and using an FC502 sterile filter (purchased from Tailin organisms, Zhejiang, Inc.), respectivelyTechnical limited) was filtered and the auxiliary material was retained on the membrane. Respectively taking the filtrate for viable count, wherein the viable count of the filtrate filtered by a 0.45 mu m mixed cellulose ester microporous filter membrane is 0CFU/g, and the viable count and the auxiliary materials are all intercepted by the membrane. Wherein the viable count of three filtrates filtered by using 5.0 μm polyethersulfone microporous filter membrane and 5.0 μm and 8.0 μm nylon microporous filter membrane is the highest, and all the viable counts are 109CFU/g, and through weighing method comparison, the polyethersulfone millipore filter membrane auxiliary material of 5.0 μm among these membranes has the highest filtration rate. The results of the above tests were combined with the properties of different material membranes, and a 5.0 μm polyethersulfone microfiltration membrane (50 mm diameter) was selected for the experiments.
Collecting filtrate filtered by 5.0 μm polyethersulfone microporous membrane, centrifuging at 8000rpm for 5min, discarding supernatant, re-suspending with sterile distilled water, precipitating to 500 μ L, and making into viable Bacillus licheniformis capsule sample suspension (10)9CFU/mL) as a pretreated product.
Example 5 optimization of Azidopropidium Bromide (PMA) concentration
Viable Staphylococcus aureus suspensions (10) prepared in example 1 were each collected8CFU/mL) and Staphylococcus aureus suspension (10) prepared in example 28CFU/mL) of the suspension, 500. mu.L each, was placed in a different centrifuge tube, and 500. mu.L of the viable Bacillus licheniformis capsule sample suspension (10) prepared in example 4 was added9CFU/mL), centrifuging at 8000rpm for 5min, discarding supernatant, and resuspending the precipitate with sterile distilled water to 0.5 mL. Adding 400 mu g/mL of PMA aqueous solution into each centrifuge tube respectively to enable the final concentration of PMA to be 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 and 110 mu g/mL respectively, carrying out dark incubation at 20-25 ℃ for 15min after uniformly mixing, carrying out vortex uniformly mixing every 5min, exposing the centrifuge tube in a photolysis instrument (an LED lamp with emission wavelength of 65-475 nm) for 20min, and uniformly mixing every 5 min. DNA is extracted by adopting a TaKaRa bacterial genome DNA extraction kit. The concentration of the extracted DNA is measured by a BioDrop ultramicro protein nucleic acid analyzer, and the result is all between 100 ng/mL and 1000 ng/mL. Then qPCR detection is carried out to obtain Ct value. The qPCR reaction system, primer probes and reaction conditions were the same as in example 3.
As is clear from FIG. 2, the rate of inhibition of the growth of dead bacteria gradually increased with the increase in the concentration of PMA. When the concentration of PMA reaches 40 mu g/mL, the Ct value is 31.416, the Ct value is basically not changed after 40 mu g/mL (P is more than 0.05, and no significant difference exists), and the inhibition rate of PMA on dead bacteria is about 99% (the average value of the Ct values of 8 points of 40 mu g/mL-110 mu g/mL is 31.770, and the inhibition rate is 31.416/31.770 is 98.9%).
As can be seen from FIG. 3, the Ct value of viable bacteria amplification at first is unchanged with increasing PMA concentration, and gradually increases when the PMA concentration exceeds 60. mu.g/mL, indicating that the PMA can significantly inhibit qPCR amplification of viable bacteria at this concentration, and the inhibition effect is more significant at higher concentrations.
Therefore, when the concentration of PMA is 40 mug/mL, the combination of PMA and the dead bacteria of the bacillus licheniformis in the sample can be deducted, the amplification of the dead bacteria DNA of the staphylococcus aureus can be effectively inhibited, and the PCR amplification of the live bacteria DNA of the staphylococcus aureus can not be influenced. 40 mug/mL is selected as the optimal working concentration of PMA.
Example 6 optimization of dark incubation time of Azidopropidium bromide
The dead staphylococcus aureus suspension (10) prepared in example 2 was collected8CFU/mL) 500. mu.L into 1.5mL centrifuge tubes, 500. mu.L of viable Bacillus licheniformis capsule sample suspension (10. mu.L) prepared in example 4 was added to each centrifuge tube9CFU/mL), 8000rpm for 5min, discarding the supernatant, and resuspending the precipitate with sterile distilled water to 0.5 mL. Adding 400 mu g/mL PMA aqueous solution into each centrifuge tube respectively to enable the final concentration to be 40 mu g/mL, carrying out vortex mixing, 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 every 5min, placing the centrifuge tubes on a photolysis instrument for exposure for 20min, and carrying out vortex mixing every 5 min. The qPCR analysis was then performed after extraction of genomic DNA using the method of example 5. The qPCR reaction system, primer probes and reaction conditions were the same as in example 3.
As can be seen from fig. 4, the Ct value did not change significantly with increasing dark incubation time, and 10min was selected as the dark incubation time in view of the durability of the method.
Example 7 optimization of Exposure time to Azide Propidium Bromide
500. mu.L of the dead Staphylococcus aureus suspension prepared in example 2 was collected(108CFU/mL) in 1.5mL centrifuge tubes, and 500. mu.L of viable Bacillus licheniformis capsule sample suspension (10) prepared in example 4 was added to each centrifuge tube9CFU/mL), 8000rpm for 5min, discarding the supernatant, and resuspending the precipitate with sterile distilled water to 0.5 mL. Adding 400 mu g/mL PMA aqueous solution into each centrifuge tube respectively to make the final concentration of 40 mu g/mL PMA aqueous solution, performing vortex mixing, performing dark incubation at 20-25 ℃ for 10min respectively, performing vortex mixing every 5min, performing exposure on a photolysis instrument for 0, 5, 10, 15, 20, 25 and 30min respectively, and performing vortex mixing every 5 min. qPCR analysis was performed after extraction of genomic DNA using the method of example 4. The qPCR reaction system, primer probes and reaction conditions were the same as in example 3.
As can be seen from FIG. 5, the Ct value gradually increases with the increase of the exposure time, and the plateau period is entered after the exposure time increases to 5 min. Considering the durability of the method and the fact that the live bacillus licheniformis capsule sample contains a certain amount of dead bacteria, the PMA and the dead bacteria are effectively combined, and 10min is selected as the exposure time.
Example 8 validation of dead and viable Staphylococcus aureus at various concentrations
Dead staphylococcus aureus suspension prepared in example 2 (10)8CFU/mL) was added to the viable bacterial suspension of Staphylococcus aureus prepared in example 1 to prepare a viable bacterial suspension of Staphylococcus aureus with viable bacterial ratios of 0%, 1%, 10%, 25%, 50%, 100%, 500. mu.L of the viable bacterial suspension of Staphylococcus aureus was placed in 1.5mL centrifuge tubes, and 500. mu.L of the viable bacterial capsule sample suspension of Bacillus licheniformis (10. mu.L) prepared in example 4 was added to each centrifuge tube9CFU/mL), 8000rpm for 5min, discarding the supernatant, and resuspending the precipitate with sterile distilled water to 0.5 mL. Adding 400 mu g/mL PMA aqueous solution into each centrifuge tube respectively to make the final concentration of 40 mu g/mL PMA aqueous solution, performing vortex mixing, performing dark incubation at 20-25 ℃ for 10min respectively, performing vortex mixing every 5min, exposing on a photolysis instrument for 10min respectively, and performing vortex mixing every 5 min. Genomic DNA was extracted for qPCR detection using the method of example 4. The qPCR reaction system, primer probes and reaction conditions were the same as in example 3.
Under the same conditions, no PMA was added as a control.
As is clear from FIG. 6, the Ct value of the experimental group without PMA remained constant as the viable cell ratio increased. In the experimental group added with PMA, the Ct value is reduced along with the increase of the viable bacteria ratio and is higher than that of the sample without PMA treatment. When the viable bacteria ratio is 100%, there is no difference in Ct values between the sample without PMA treatment and the sample with PMA treatment. The experimental conditions established by the method show that PMA can be effectively combined with dead bacteria DNA, so that the subsequent DNA amplification is inhibited, false positive results are avoided, the detection of the live bacteria is not influenced, and false negative results are avoided.
Example 9 sensitivity study
10 prepared by the method of example 1 was dissolved in physiological saline8Sequentially carrying out gradient dilution by 10 times on the CFU/mL staphylococcus aureus viable bacteria suspension to obtain 10-fold concentration8、107、106、105、104、103、102、101、100500. mu.L of CFU/mL bacterial solution was placed in 1.5mL centrifuge tubes, and 0.5mL of the suspension of viable Bacillus licheniformis capsules (10) prepared according to example 4 was added to each centrifuge tube9CFU/mL), 8000rpm for 5min, discarding the supernatant, and respectively resuspending the precipitate with sterile distilled water to 0.5 mL. Adding 400 mu g/mL PMA aqueous solution into each centrifuge tube respectively to make the final concentration of 40 mu g/mL PMA aqueous solution, performing vortex mixing, performing dark incubation at 20-25 ℃ for 10min respectively, performing vortex mixing every 5min, exposing on a photolysis instrument for 10min respectively, and performing vortex mixing every 5 min. Genomic DNA was extracted for qPCR detection using the method of example 4. The qPCR reaction system, primer probes and reaction conditions were the same as in example 3.
As can be seen in FIG. 7, the concentration of viable bacterial suspension in Staphylococcus aureus was 104～108Shows good linear relation with Ct value in the CFU/mL range (R)20.9916), the standard curve is-2.8905 x +34.025, and the detection limit is 104CFU/mL。
Example 10 specificity study
1mL of the viable staphylococcus aureus suspension and 1mL of the viable staphylococcus epidermidis suspension prepared in the example 1 are respectively put into 2mL centrifuge tubes, and 500 mu L of the viable bacillus licheniformis capsule sample prepared in the example 4 is respectively added into each centrifuge tubeSuspension (10)9CFU/mL), 8000rpm for 5min, discarding the supernatant, and resuspending the precipitate with sterile distilled water to 0.5 mL. Adding 400 mu g/mL PMA aqueous solution into each centrifuge tube respectively to make the final concentration of 40 mu g/mL PMA aqueous solution, performing vortex mixing, performing dark incubation at 20-25 ℃ for 10min respectively, performing vortex mixing every 5min, exposing on a photolysis instrument for 10min respectively, and performing vortex mixing every 5 min. qPCR analysis was performed after extraction of genomic DNA using the method of example 4. The qPCR reaction system, primer probes and reaction conditions were the same as in example 3.
The exponential phase of the fluorescence amplification signal of staphylococcus aureus is obvious, the Ct value is 16.312, and as can be seen from figure 8, no obvious fluorescence amplification curve is seen in staphylococcus epidermidis, which shows that the method has high detection specificity on staphylococcus aureus.
Sequence listing
<110> Zhejiang province food and drug inspection research institute
<120> method for detecting staphylococcus aureus in microecological viable bacteria product
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<213> Unknown (Unknown)
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<212> DNA
<213> Unknown (Unknown)
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Claims (8)
1. A method for detecting staphylococcus aureus in a microecological live bacterial product is characterized by comprising the following steps:
(1) sample pretreatment: adding the viable bacteria product into sterile physiological saline, homogenizing, filtering with polyethersulfone microporous filter membrane, collecting filtrate, centrifuging, discarding supernatant, and resuspending the precipitate with sterile distilled water to obtain pretreated viable bacteria product resuspension;
(2) pre-treating azide propidium bromide: adding azide propidium bromide into the live bacteria product heavy suspension pretreated in the step (1), fully and uniformly 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 pretreated live bacteria product treatment liquid;
(3) real-time fluorescent quantitative PCR detection:
extracting the genome DNA of the viable bacteria product pretreated in the step (2) as a template, carrying out qPCR reaction to obtain a Ct value, and when the Ct value is less than 31, determining viable staphylococcus aureus in the microbial viable bacteria product to be detected;
an upstream primer: 5'-TTCTTCACGACTAAATAAACGCTC A-3', respectively;
a downstream primer: 5 'GGTACTACTAAAGATTATCAAGACGGCT-3';
the probe sequence is as follows: 5 '-ROX-CAGAACACAATGTTTCCGATGCAACGT-BHQ 2-3'.
2. The method according to claim 1, wherein in the step (1), the volume of the sterile physiological saline is 12-57-ml/3g based on the weight of the viable bacteria preparation.
3. The method according to claim 1, wherein in step (1), the pore size of the polyethersulfone microfiltration membrane is 3.0-6.0 μm.
4. The method of claim 1, wherein in step (2), said azido propidium bromide is added as a 400 μ g/mL aqueous solution; the final concentration of the azide propidium bromide is 30-60 mug/mL.
5. The method as claimed in claim 1, wherein in step (2), the emission wavelength of the LED lamp is 465-475 nm.
6. The method of claim 1, wherein in step (2), the LED lamp is exposed for 15min and mixed evenly every 5 min.
7. The method of claim 1, wherein in step (3), the qPCR reaction system: ProbeqPCR Mix (with UNG) 10. mu.L; the upstream primer and the downstream primer are respectively 0.4 mu L; probe 0.8 μ L; 2 mu L of template DNA; ddH2O6.2. mu.L, total volume 20. mu.L.
8. The method of claim 1, wherein in step (3), the qPCR reaction conditions: preheating at 25 deg.C for 10 min; stage1 pre-denaturation: at 95 ℃ for 30 s; stage2PCR reaction: 95 ℃,5s, 60 ℃,34s, 40 cycles.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104726576A (en) * | 2015-03-13 | 2015-06-24 | 上海交通大学 | Method for detecting staphylococcus aureus and enterotoxin A gene in food |
| CN104726550A (en) * | 2014-11-11 | 2015-06-24 | 南昌大学 | Kit for detecting staphylococcus aureus viable cells in food |
| CN105612254A (en) * | 2013-10-07 | 2016-05-25 | 三井化学株式会社 | Pcr primer set for bacterial dna amplification, kit for detecting and/or identifying bacterial species, and method for detecting and/or identifying bacterial species |
| CN108018366A (en) * | 2018-01-04 | 2018-05-11 | 南京农业大学 | Reagent, kit and application for staphylococcus aureus living stems |
| CN109735599A (en) * | 2019-01-18 | 2019-05-10 | 河南省商业科学研究所有限责任公司 | A method of improving staphylococcus aureus detection limit in food liquid easy to foaming |
| CN111235236A (en) * | 2020-02-15 | 2020-06-05 | 新疆农业大学 | Method for rapidly detecting viable bacteria of erwinia amylovora and application |
-
2020
- 2020-11-23 CN CN202011318149.6A patent/CN112301140B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105612254A (en) * | 2013-10-07 | 2016-05-25 | 三井化学株式会社 | Pcr primer set for bacterial dna amplification, kit for detecting and/or identifying bacterial species, and method for detecting and/or identifying bacterial species |
| CN111534623A (en) * | 2013-10-07 | 2020-08-14 | 三井化学株式会社 | Primer set used in PCR for amplifying bacterial DNA, kit and method thereof |
| CN104726550A (en) * | 2014-11-11 | 2015-06-24 | 南昌大学 | Kit for detecting staphylococcus aureus viable cells in food |
| CN104726576A (en) * | 2015-03-13 | 2015-06-24 | 上海交通大学 | Method for detecting staphylococcus aureus and enterotoxin A gene in food |
| CN108018366A (en) * | 2018-01-04 | 2018-05-11 | 南京农业大学 | Reagent, kit and application for staphylococcus aureus living stems |
| CN109735599A (en) * | 2019-01-18 | 2019-05-10 | 河南省商业科学研究所有限责任公司 | A method of improving staphylococcus aureus detection limit in food liquid easy to foaming |
| CN111235236A (en) * | 2020-02-15 | 2020-06-05 | 新疆农业大学 | Method for rapidly detecting viable bacteria of erwinia amylovora and application |
Non-Patent Citations (1)
| Title |
|---|
| 柯振华: "餐饮食品中6 病原菌叠氮丙啶-定量聚合酶链反应检测方法开发与检验数据分析" * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113046451A (en) * | 2021-03-19 | 2021-06-29 | 厦门承葛医学检验实验室有限公司 | Live bacterium quantification method of flora and application thereof |
| CN113046451B (en) * | 2021-03-19 | 2022-08-23 | 厦门承葛医学检验实验室有限公司 | Live bacterium quantification method of flora and application thereof |
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