WO2022141945A1 - Detection method for live bacteria of standard strain of food-borne pathogenic bacteria having specific molecular target, and use - Google Patents

Detection method for live bacteria of standard strain of food-borne pathogenic bacteria having specific molecular target, and use Download PDF

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WO2022141945A1
WO2022141945A1 PCT/CN2021/087080 CN2021087080W WO2022141945A1 WO 2022141945 A1 WO2022141945 A1 WO 2022141945A1 CN 2021087080 W CN2021087080 W CN 2021087080W WO 2022141945 A1 WO2022141945 A1 WO 2022141945A1
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primers
amplification
nucleotide sequence
set forth
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吴清平
张菊梅
丁郁
薛亮
陈谋通
相欣然
王涓
叶青华
吴诗
曾海燕
杨小鹃
张淑红
庞锐
雷涛
古其会
韦献虎
张友雄
陈鲁
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广东省科学院微生物研究所(广东省微生物分析检测中心)
广东环凯生物科技有限公司
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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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the technical field of microorganism detection, and in particular relates to a detection method for standard strain viable bacteria of food-borne pathogenic bacteria and primers for specific molecular targets thereof.
  • Foodborne pathogens are the main cause of food-borne illnesses.
  • Foodborne pathogens mainly include: DiarrhoeagenicEscherichiacoli, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus , Cronobacterspp., Bacillus cereus, Salmonella spp., Yersinia enterocolitica and Campylobacter jejuni.
  • ATCC American Type Culture Collection
  • CMCC my country Medical Microorganisms Center
  • the standard strains of food-borne pathogenic bacteria selected by the present invention are all dominant isolates with typical biochemical characteristics in food samples contaminated in China, and can better reflect the genetics of food-borne pathogenic bacteria in food-derived isolates in China. background.
  • the establishment of a live cell quantitative detection method for the standard strains of food-borne pathogens can realize the evaluation of the preservation performance of the standard strains, and provide an important guarantee for the production and application of the standard strains.
  • PCR polymerase chain reaction
  • qPCR Real-time quantitative PCR
  • PMA Propidium monoazide
  • PCR polymerase chain reaction
  • the plate culture method and the nucleic acid amplification method of conventional primers lack the specific identification ability of food-borne standard strains, and it is difficult to realize the identification at the strain level in the evaluation of the preservation performance of standard strains. Therefore, the establishment of a live cell quantitative detection method using the strain-specific gene sequences of food-borne pathogen standard strains is of great significance for the rapid confirmation of the source of food-borne pathogen strains and the efficient evaluation of the preservation quality of the strains.
  • the invention provides a kind of detection method of food-borne pathogenic bacteria viable bacteria, the method is specially designed for the viable bacteria detection method of the bacterial strains shown in Table 1 with the preservation number, because these bacterial strains have respectively A specific molecular target can be detected by specific primers. Therefore, the present invention is used to detect the number of viable cells of each of the above-mentioned strains by combining the PMA-qPCR detection method with the primers of the specific molecular targets of the above-mentioned strains.
  • the present invention provides specific molecular targets for detecting food-borne pathogens, the nucleotide sequences of which are shown in SEQ ID NOs: 1-31.
  • the specific molecular targets are detected using primers whose nucleic acid sequences are shown in SEQ ID NOs: 32-93, respectively.
  • the food-borne pathogenic bacteria include Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Yersinia enterocolitica, Bacillus cereus, Cronobacter, diarrhoea Escherichia coli, Salmonella and Campylobacter jejuni.
  • the present invention also provides a detection method for food-borne pathogenic bacteria viable bacteria, comprising the following steps:
  • Step 1 Add PMA dye solution to the sample to be tested
  • Step 2 Extract the bacterial DNA in the sample to be tested
  • Step 3 use the bacterial DNA in step 2 as a template to perform real-time fluorescence quantitative PCR detection
  • Step 4 After the reaction is completed, calculate the number of viable food-borne pathogenic bacteria in the sample by comparing the obtained curve and Ct value of fluorescence quantitative PCR amplification with the established standard curve;
  • the primers used for real-time fluorescence quantitative PCR detection in the step 3 include nucleic acid sequences as shown in SEQ ID NOs: 32-93.
  • the nucleotide sequence of the target fragment detected and amplified by real-time fluorescence quantitative PCR is shown in SEQ ID NOs: 1-31.
  • the PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:1 include: an upstream primer shown in SEQ ID NO:32 and a downstream primer shown in SEQ ID NO:33; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:2 include: upstream primers as shown in SEQ ID NO:34 and downstream primers as shown in SEQ ID NO:35;
  • the PCR primers for amplification of the nucleotide sequence shown in: 3 include the upstream primer shown in SEQ ID NO: 36 and the downstream primer shown in SEQ ID NO: 37; for the nucleus shown in SEQ ID NO: 4
  • the PCR primers for nucleotide sequence amplification include upstream primers as shown in SEQ ID NO: 38 and downstream primers as shown in SEQ ID NO: 39; for the nucleotide sequence amplification as shown in SEQ ID NO: 5
  • PCR primers include upstream primers as shown in SEQ ID NO:40
  • the food-borne pathogenic bacteria include at least one of the following bacteria species: Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Yersinia enterocolitica, Bacillus cereus Bacillus, Cronobacter, diarrhea-causing Escherichia coli, Salmonella, Campylobacter jejuni.
  • the concentration of foodborne pathogenic bacteria in the sample to be tested in the step 1 is controlled within the range of 1 ⁇ 10 4 to 1 ⁇ 10 6 cfu/mL.
  • step 1 the specific operations of step 1 are as follows: add PMA solution to the sample to be tested, place it in a 37°C constant temperature incubator for 5 minutes in the dark, and irradiate it with a 500W tungsten lamp for 10 minutes.
  • the purpose of this step is to cross-link the DNA of dead bacteria or bacteria with damaged cell membranes to PMA so that PCR amplification cannot be performed.
  • the final concentration of PMA is 2-16 mg/mL. More preferably, the final concentration of PMA is 16 mg/mL.
  • the present invention finally obtains the DNA that can inhibit the above-mentioned food-borne pathogenic and dead bacteria at the same time under the condition of this concentration through optimization.
  • the bacterial cell DNA is extracted by magnetic bead method to extract the DNA in the bacterial suspension.
  • the reaction system of real-time fluorescence quantitative PCR in the step 3 is: 10 ⁇ L of 2 ⁇ qPCR reaction buffer, 0.4 ⁇ L of 5 ⁇ M upstream primer, 0.4 ⁇ L of 5 ⁇ M downstream primer, 2 ⁇ L of DNA template, 7.2 ⁇ L of sterilized double-distilled water, and the total Volume 20 ⁇ L.
  • the reaction procedure of real-time fluorescence quantitative PCR in the step 3 is: (1) pre-denaturation: 95°C, 30s, 20°C/s, 1Cycle; (2) PCR amplification reaction: 95°C, 5s, 20°C /s, 60°C, 5s, 20°C/s, 40Cycles; (3) Dissolution curve analysis: 95°C, 0s, 20°C/s, 65°C, 15s, 20°C/s, 95°C, 0s, 0.1°C/ s.
  • the present invention discloses 40 specific gene targets and related PMA-qPCR quantitative detection methods for identifying food-borne pathogenic bacteria;
  • the method has the advantages of short detection time, does not need to go through the traditional culture and enrichment process, and is not interfered by the dead bacteria of the standard food-borne pathogenic bacteria in the sample, and can accurately detect and quantify the specific bacteria quantity of the standard food-borne pathogenic bacteria in the sample. , to effectively avoid false positive results.
  • the selected molecular targets are the unique nucleic acid sequences of each food-borne standard strain.
  • the detection results are highly specific and have no cross-reaction with other closely related and homologous strains. The results are simple to determine, and the detection results are basically consistent with the traditional plate counting method.
  • Figure 1 shows the qPCR results for the evaluation of the effect of PMA on the DNA amplification of dead bacteria and the electrophoresis of standard strains treated with different concentrations of PMA: (1) 2-16 are the results of gel running after treatment with concentrations of 2-16 ⁇ g/mL PMA, M is 2000Maker, (2 ) qPCR results of PMA-treated and untreated samples containing dead bacteria and samples containing viable bacteria;
  • Figure 2 shows the results of PCR electrophoresis of PMA-treated and untreated food-borne pathogenic bacteria samples with different concentration gradients
  • Fig. 3 is the inhibitory effect of PMA treatment method on the dead bacteria of various food-borne pathogens
  • Figure 4 is a standard curve for the detection of foodborne Escherichia coli live cells
  • Fig. 5 is the standard curve of live cell detection of foodborne Salmonella
  • Figure 6 is the standard curve of foodborne Cronobacter live cell detection
  • Figure 7 is a standard curve for the detection of food-borne Vibrio parahaemolyticus viable cells
  • Fig. 8 is the standard curve of live cell detection of foodborne Listeria monocytogenes
  • Fig. 9 is the standard curve of live cell detection of foodborne Bacillus cereus
  • Figure 10 is a standard curve for the detection of foodborne Yersinia enterocolitica live cells
  • Figure 11 is the standard curve for the detection of food-borne Campylobacter jejuni live cells
  • Figure 12 is the standard curve for the detection of food-borne Staphylococcus aureus live cells.
  • Table 1 Specific molecular targets and primers for each foodborne pathogen
  • Step 1 Add PMA dye solution to the sample to be tested, the specific operation is as follows;
  • Step 2 Extract the bacterial DNA in the sample to be tested, and the specific operations are as follows;
  • the bacterial suspension NDA was extracted by the magnetic bead method, and the treated bacterial suspension was centrifuged at 10,000 r/min, the supernatant was removed as much as possible, and 180 ⁇ L LTE buffer containing lysozyme was added to the precipitate, and the system was fully mixed by vortexing. , 37 °C water bath (60min for Gram-positive bacteria, 30min for Gram-negative bacteria).
  • Step 3 Use the bacterial DNA in Step 2 as a template to perform real-time fluorescence quantitative PCR detection, and the specific operations are as follows;
  • the specific nucleic acid sequences of each food-borne pathogenic bacteria are quantitatively detected according to the primers designed for each specific primer and the fluorescent dye intercalation method (q-PCR) according to the above-mentioned table 1, and the amplification parameters are specifically:
  • the qPCR reaction system is:
  • the qPCR reaction program is:
  • Step 4 After the above reaction is completed, calculate the number of viable food-borne pathogenic bacteria in the sample by comparing the obtained curve and Ct value of fluorescence quantitative PCR amplification with the established standard curve; the specific operations are as follows:
  • Strain PY002 has an R2 of 0.97924 with a detection limit of 10 2 cfu/mL; strain 2968A1 has an R2 of 0.99747 with a detection limit of 10 2 cfu/mL; strain 3164A1 has an R2 of 0.97441 with a detection limit of 10 3 cfu/mL; strain 3466A3
  • the R2 of strain 3025B1 was 0.98487, and the detection limit was 10 2 cfu/mL; the R2 of strain 3025B1 was 0.98813, and the detection limit was 10 2 cfu/mL; the R2 of strain 3776A3-1 was 0.99487, and the detection limit was 10 2 cfu/mL.
  • the R2 of strain FSCC(I)215032 was 0.98151, and the detection limit was 10 2 cfu/mL; the R2 of strain FSCC(I)21501206 was 0.9767, and the detection limit was 10 2 cfu/mL; the R2 of strain FSCC(I) 215467 was 0.99947 , the detection limit was 10 2 cfu/mL; the R2 of strain FSCC(I)215456 was 0.98519, and the detection limit was 10 2 cfu/mL; the R2 of strain FSCC(I) 215032 was 0.99634, and the detection limit was 10 2 cfu/mL.
  • the R2 of strain cro359W is 0.9964, and the limit is 10 2 cfu/mL; the R2 of strain cro509C1 is 0.99483, and the detection limit is 10 3 cfu/mL; the R2 of strain cro611A3 is 0.98703, and the detection limit is 10 2 cfu/mL; The R2 was 0.997, and the detection limit was 10 4 cfu/mL; the R2 of the strain cro1537W was 0.99424, and the detection limit was 10 3 cfu/mL.
  • the R2 of strain VP2227C2 was 0.99359, and the detection limit was 10 3 cfu/mL; the R2 of strain VPS179C3 was 0.98814, and the detection limit was 10 3 cfu/mL; the R2 of strain 3630A3 was 0.96913, and the detection limit was 10 2 cfu/mL.
  • Strain 428-1LM has an R2 of 0.99454 with a detection limit of 10 3 cfu/mL; strain 615-1LM has an R2 of 0.9695 with a detection limit of 10 2 cfu/mL; standard strain 678-1LM has an R2 of 0.98931 with a detection limit of 10 4 cfu/mL; strain 833-1LM had an R2 of 0.99363 with a detection limit of 10 3 cfu/mL; strain 1382-1LM had an R2 of 0.95412 with a detection limit of 10 3 cfu/mL.
  • the R2 of strain 260-1B was 0.97443, and the detection limit was 10 3 cfu/mL; the R2 of strain Y1712 was 0.98046, and the detection limit was 10 3 cfu/mL; the R2 of strain 1761-2A was 0.99759, and the detection limit was 10 3 cfu/mL. mL; the R2 of strain 2801 was 0.99613, and the detection limit was 10 2 cfu/mL;
  • the R2 of strain 2841-1B was 0.99367 with a detection limit of 103 cfu/mL.
  • the R2 of strain c009 was 0.99942, and the detection limit was 10 2 cfu/mL; the R2 of strain y802 was 0.99615, and the detection limit was 10 2 cfu/mL; the R2 of strain c1702 was 0.9906, and the detection limit was 10 1 cfu/mL.
  • the R2 of strain GDMCC 60857 was 0.96844 with a detection limit of 10 4 cfu/mL; the R2 of strain GDMCC 60858 was 0.96895 with a detection limit of 10 5 cfu/mL.
  • the R2 of strain Sta144-2 is 0.96381, and the detection limit is 10 2 cfu/mL; the R2 of strain Sta403 is 0.99793, and the detection limit is 10 2 cfu/mL; the R2 of strain Sta177-0 is 0.99796, and the detection limit is 10 5 cfu/mL mL; the R2 of strain Sta1942-0 was 0.99473, and the detection limit was 10 5 cfu/mL; the R2 of strain Sta370B3 was 0.99942, and the detection limit was 10 3 cfu/mL; the R2 of strain Sta4127 was 0.983, and the detection limit was 10 4 cfu/mL mL.
  • Embodiment 2 PMA-qPCR detection system PMA condition optimization
  • Salmonella FSCC(I) 215467 4 mL of 10 6 cfu/mL bacterial solution was taken and divided into 4 parts, and 2 parts were prepared as dead bacteria samples by heating. Take a sample of live bacteria and dead bacteria and treat with PMA: centrifuge to remove the supernatant, add 1 mL of deionized water to reconstitute, then add 10 ⁇ L of 1 mg/mL PMA solution, vortex for 10 s, cover with aluminum foil and shake at room temperature. Incubate with light for 5 min, and irradiate with a 500W tungsten filament lamp for 10 min to fully cross-link PMA with dead bacterial DNA.
  • the detection sensitivity decreased from 10 5 to 10 6 cfu/mL
  • the detection sensitivity of FSCC(I)21501206 decreased from 10 3 to 10 5 cfu/mL
  • the detection sensitivity of FSCC(I)215467 decreased from 10 6 to 10 7 cfu/mL. It shows that PMA-PCR method can effectively remove the interference of dead bacterial DNA in common concentration gradients and reduce false positive results in the processing of actual samples.
  • the dead bacteria suspension with the common concentration of each foodborne pathogen was prepared to detect the highest limit of the dead bacteria concentration of each strain by PMA-PCR method. The test results are shown in Figure 4.
  • the highest concentration of 16 ⁇ g/mL PMA that can effectively inhibit each standard strain is: Vibrio parahaemolyticus (10 6 cfu/mL), diarrhea-causing Escherichia coli (10 6 cfu/mL), Listeria monocytogenes (10 6 cfu/mL), Staphylococcus aureus (10 5 cfu/mL), Cronobacter (10 5 cfu/mL), Bacillus cereus (10 4 cfu/mL), Salmonella (10 6 cfu/mL) , Yersinia enterocolitica (10 6 cfu/mL), Campylobacter jejuni (10 4 cfu/mL).

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Abstract

Provided is a specific molecular target for detecting a food-borne pathogenic bacteria. The nucleotide sequence of the specific molecular target is shown in SEQ ID NOs: 1-31. Also provided in the present invention is a detection method for live bacteria of food-borne pathogenic bacteria. The method comprises adding PMA dye solution into a sample to be tested, and then carrying out real-time fluorescence quantitative PCR detection.

Description

具有特异性分子靶标的食源性致病菌标准菌株活菌的检测方法及应用Detection method and application of live bacteria of food-borne pathogenic bacteria standard strains with specific molecular targets 技术领域technical field
本发明属于微生物检测技术领域，具体涉及一种食源性致病菌标准菌株活菌的检测方法及其特异性分子靶标的引物。The invention belongs to the technical field of microorganism detection, and in particular relates to a detection method for standard strain viable bacteria of food-borne pathogenic bacteria and primers for specific molecular targets thereof.
背景技术Background technique
食源性致病菌是其引起的食物疾病的主要原因。食源性致病菌主要包括：致泻大肠埃希氏菌(DiarrhoeagenicEscherichiacoli)、单核细胞增生李斯特菌(Listeria monocytogenes)、副溶血性弧菌(Vibrio parahaemolyticus)、金黄色葡萄球菌(Staphylococcus aureus)、克罗诺杆菌(Cronobacterspp.)、蜡样芽胞杆菌(Bacillus cereus)、沙门氏菌(Salmonella spp.)、小肠结肠炎耶尔森氏菌(Yersinia enterocolitica)和空肠弯曲菌(Campylobacter jejuni)。目前对于标准菌株主要来源于国际或国内菌种保藏中心保藏的，遗传学特性得到确认和保证并可追溯，如美国微生物菌株保藏中心(American Type Culture Collection，ATCC)、我国医学微生物菌种中心(CMCC)等。本发明所选的食源性致病菌标准菌株均为污染中国地区食品样品中具有典型生化特征的优势分离株，能够较好地反映食源性致病菌在中国地区食品源分离株的遗传背景。建立针对食源性致病菌标准菌株的活细胞定量检测方法可以实现对标准菌株保藏性能的评估，为标准菌株的生产和应用提供重要保证。Foodborne pathogens are the main cause of food-borne illnesses. Foodborne pathogens mainly include: DiarrhoeagenicEscherichiacoli, Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus , Cronobacterspp., Bacillus cereus, Salmonella spp., Yersinia enterocolitica and Campylobacter jejuni. At present, the standard strains are mainly from the collection of international or domestic culture collection centers, and their genetic characteristics have been confirmed, guaranteed and traceable, such as the American Type Culture Collection (ATCC), the my country Medical Microorganisms Center ( CMCC), etc. The standard strains of food-borne pathogenic bacteria selected by the present invention are all dominant isolates with typical biochemical characteristics in food samples contaminated in China, and can better reflect the genetics of food-borne pathogenic bacteria in food-derived isolates in China. background. The establishment of a live cell quantitative detection method for the standard strains of food-borne pathogens can realize the evaluation of the preservation performance of the standard strains, and provide an important guarantee for the production and application of the standard strains.
在对实际样品检测中致病菌检测和菌种保藏质量评估方面，目前普遍采用基于传统培养的平板计数法。该方法需要通过梯度稀释、选择性培养基培养等一系列繁琐步骤。目前已开发多种基于特异性核酸序列的快速检测方法如聚合酶链式反应(Polymerase chain reaction,PCR)已被视为检测食品中食源性致病菌的可靠方法，实时荧光定量PCR(qPCR)由于其特异性和灵敏性，可以克服常规生物学方法的这些缺点，并被广泛应用于食源性病原菌快速检测，其扩增的核酸序列基本采用菌株所在属种常见毒力基因进行扩增鉴定。In the detection of pathogenic bacteria in the detection of actual samples and the quality assessment of bacterial species preservation, the plate counting method based on traditional culture is generally used. This method requires a series of tedious steps such as gradient dilution and selective medium cultivation. At present, a variety of rapid detection methods based on specific nucleic acid sequences have been developed, such as polymerase chain reaction (PCR), which has been regarded as a reliable method for the detection of food-borne pathogens in food. Real-time quantitative PCR (qPCR) ) due to its specificity and sensitivity, it can overcome these shortcomings of conventional biological methods, and is widely used in the rapid detection of food-borne pathogens. identification.
叠氮溴化丙啶(Propidium monoazide,PMA)是一种对核酸具有高度亲和力的光敏反应染料，其能穿透死细胞和受损细胞的细胞膜，在强光下与DNA形成共价键，从而抑制死细胞中DNA的扩增，而活细胞的DNA未被结合，可在qPCR反应中被扩增和检测，以达到区分死、活菌检测的目的。近年来，利用PMA结合DNA扩增的技术被广泛应用于多种食源性致病菌的活菌检测，被认为是目前最为有效的新型活菌检测技术。Propidium monoazide (PMA) is a photosensitive dye with high affinity for nucleic acids, which can penetrate the cell membranes of dead and damaged cells and form covalent bonds with DNA under strong light, thereby Inhibit the amplification of DNA in dead cells, while the DNA of living cells is not bound, and can be amplified and detected in the qPCR reaction to achieve the purpose of distinguishing between dead and live bacteria. In recent years, the technology using PMA combined with DNA amplification has been widely used in the detection of live bacteria of a variety of food-borne pathogens, and is considered to be the most effective new live bacteria detection technology at present.
目前，针对样品中菌株活细胞定量仍然普遍采用国标规定的传统培养方法后进行平板计数的方法，该方法结果较为准确，检测时间较长(一周左右)、操作复杂(预增菌、选择性培养、显色培养、生化鉴定)、成本高，且灵敏度较低，特异性较差。目前已开发多种基于特异性核酸序列的快速检测方法如聚合酶链式反应(Polymerase chain reaction,PCR)已被视为检测乳或其他食品中食源性致病菌的可靠方法，实时荧光定量PCR(qPCR)由于其特异性和灵敏性，则可以克服常规生物学方法的这些缺点。然而，使用qPCR难以区分活细胞和死细胞，这导致了活细胞数量可能会被高估，存在假阴性和假阳性的问题。At present, the traditional culture method stipulated by the national standard is still widely used for the quantification of viable cells in the sample, and then the plate counting method is generally used. , chromogenic culture, biochemical identification), high cost, low sensitivity and poor specificity. At present, a variety of rapid detection methods based on specific nucleic acid sequences have been developed, such as polymerase chain reaction (PCR), which has been regarded as a reliable method for the detection of food-borne pathogens in milk or other foods. PCR (qPCR) can overcome these shortcomings of conventional biological methods due to its specificity and sensitivity. However, it is difficult to distinguish live cells from dead cells using qPCR, which leads to the possible overestimation of the number of live cells and the problem of false negatives and false positives.
此外，平板培养法和常规引物的核酸扩增方法缺乏对食源性标准菌株的特异性识别能力，在对标准菌株保藏性能评估难以实现菌株水平上的鉴定。因此，利用食源性致病菌标准菌株的菌株特异性基因序列的活细胞定量检测方法的建立对于食源性病原菌菌株来源的快速确证和菌种保藏质量的高效评估具有重要意义。In addition, the plate culture method and the nucleic acid amplification method of conventional primers lack the specific identification ability of food-borne standard strains, and it is difficult to realize the identification at the strain level in the evaluation of the preservation performance of standard strains. Therefore, the establishment of a live cell quantitative detection method using the strain-specific gene sequences of food-borne pathogen standard strains is of great significance for the rapid confirmation of the source of food-borne pathogen strains and the efficient evaluation of the preservation quality of the strains.
发明内容SUMMARY OF THE INVENTION
为解决上述技术问题，本发明提供了一种食源性致病菌活菌的检测方法，该方法专门针对保藏编号分别如表1所示的菌株设计的活菌检测方法，由于这些菌株分别具有一段特异性的分子靶标，可通过特异性的引物进行检测。因此，本发明是通过将PMA-qPCR检测方法与上述各菌株的特异性分子靶标的引物结合用于检测上述各个菌株的活细胞数量。In order to solve the above-mentioned technical problems, the invention provides a kind of detection method of food-borne pathogenic bacteria viable bacteria, the method is specially designed for the viable bacteria detection method of the bacterial strains shown in Table 1 with the preservation number, because these bacterial strains have respectively A specific molecular target can be detected by specific primers. Therefore, the present invention is used to detect the number of viable cells of each of the above-mentioned strains by combining the PMA-qPCR detection method with the primers of the specific molecular targets of the above-mentioned strains.
本发明通过以下技术方案实现：The present invention is achieved through the following technical solutions:
本发明提供了用于检测食源性致病菌的特异性分子靶标，其核苷酸序列如SEQ ID NO:1～31所示。The present invention provides specific molecular targets for detecting food-borne pathogens, the nucleotide sequences of which are shown in SEQ ID NOs: 1-31.
优选地，所述特异性分子靶标使用核酸序列如SEQ ID NO：32～93所示的引物分别检测。Preferably, the specific molecular targets are detected using primers whose nucleic acid sequences are shown in SEQ ID NOs: 32-93, respectively.
优选地，所述食源性致病菌包括单增李斯特菌、副溶血性弧菌、金黄色葡萄球菌、小肠结肠炎耶尔森氏菌、蜡样芽孢杆菌、克罗诺杆菌、致泻大肠埃希氏菌、沙门氏菌和空肠弯曲杆菌。Preferably, the food-borne pathogenic bacteria include Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Yersinia enterocolitica, Bacillus cereus, Cronobacter, diarrhoea Escherichia coli, Salmonella and Campylobacter jejuni.
本发明还提供了一种食源性致病菌活菌的检测方法，包括以下步骤：The present invention also provides a detection method for food-borne pathogenic bacteria viable bacteria, comprising the following steps:
步骤1：在待测样品加入PMA染料溶液；Step 1: Add PMA dye solution to the sample to be tested;
步骤2：提取待测样品中的菌体DNA；Step 2: Extract the bacterial DNA in the sample to be tested;
步骤3：以步骤2中的菌体DNA为模板，进行实时荧光定量PCR检测；Step 3: use the bacterial DNA in step 2 as a template to perform real-time fluorescence quantitative PCR detection;
步骤4：待反应结束后，将获得的荧光定量PCR扩增的曲线和Ct值与建立的标准曲线计算样品中食源性致病菌活菌的数量；Step 4: After the reaction is completed, calculate the number of viable food-borne pathogenic bacteria in the sample by comparing the obtained curve and Ct value of fluorescence quantitative PCR amplification with the established standard curve;
其中，所述步骤3中进行实时荧光定量PCR检测所使用的引物包括如SEQ ID NO：32～93所示的核酸序列。Wherein, the primers used for real-time fluorescence quantitative PCR detection in the step 3 include nucleic acid sequences as shown in SEQ ID NOs: 32-93.
优选地，所述步骤2中实时荧光定量PCR检测扩增的目的片段的核苷酸序列如SEQ ID NO：1～31所示。Preferably, in the step 2, the nucleotide sequence of the target fragment detected and amplified by real-time fluorescence quantitative PCR is shown in SEQ ID NOs: 1-31.
优选地，所述针对如SEQ ID NO:1所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:32所示的上游引物和如SEQ ID NO:33所示的下游引物；针对如SEQ ID NO:2所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:34所示的上游引物和如SEQ ID NO:35所示的下游引物；针对如SEQ ID NO:3所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:36所示的上游引物和如SEQ ID NO:37所示的下游引物；针对如SEQ ID NO:4所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:38所示的上游引物和如SEQ ID NO:39所示的下游引物；针对如SEQ ID NO:5所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:40所示的上游引物和如SEQ ID NO:41所示的下游引物；针对如SEQ ID NO:6所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:42所示的上游引物和如SEQ ID NO:43所示的下游引物；针对如SEQ ID NO:7所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:44所示的上游引物和如SEQ ID NO:45所示的下游引物；针对如SEQ ID NO:8所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:46所示的上游引物和如SEQ ID NO:47所示的下游引物；针对如SEQ ID NO:9所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:48所示的上游引物和如SEQ ID NO:49所示的下游引物；针对如SEQ ID NO:10所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:50所示的上游引物和如SEQ ID NO:51所示的下游引物；针对如SEQ ID NO:11所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:52所示的上游引物和如SEQ ID NO:53所示的下游引物；优选地，所述针对如SEQ ID NO:12所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:54所示的上游引物和如SEQ ID NO:55所示的下游引物；针对如SEQ ID NO:13所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:56所示的上游引物和如SEQ ID NO:57所示的下游引物；针对如SEQ ID NO:14所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:58所示的上游引物和如SEQ ID NO:59所示的下游引物；针对如SEQ ID NO:15所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:60所示的上游引物和如SEQ ID NO:61所示的下游引物；针对如SEQ ID NO:16所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:62所示的上游引物和如SEQ ID NO:63所示的下游引物；针对如SEQ ID NO:17所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:64所示的上游引物和如SEQ ID NO:65所示的下游引物；针对如SEQ ID NO:18所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:66所示的上游引物和如SEQ ID NO:67所示的下游引物；针对如SEQ ID NO:19所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:68所示的上游引物和如SEQ ID NO:69所示的下游引物；针对如SEQ ID NO:20所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:70所示的上游引物和如SEQ ID NO:71所示的下游引物；针对如SEQ ID NO:21所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:72所示的上游引物和如SEQ ID NO:73所示的下游引物；针对如SEQ ID NO:22所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:74所示的上游引物和如SEQ ID NO:75所示的下游引物；所述针对如SEQ ID NO:23所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:76所示的上游引物和如SEQ ID NO:77所示的下游引物；针对如SEQ ID NO:24所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:78所示的上游引物和如SEQ ID NO:79所示的下游引物；针对如SEQ ID NO:25所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:80所示的上游引物和如SEQ ID NO:81所示的下游引物；针对如SEQ ID NO:26所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:82所示的上游引物和如SEQ ID NO:83所示的下游引物；针对如SEQ ID NO:27所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:84所示的上游引物和如SEQ ID NO:85所示的下游引物；针对如SEQ ID NO:28所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:86所示的上游引物和如SEQ ID NO:87所示的下游引物；针对如SEQ ID NO:29所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:88所示的上游引物和如SEQ ID NO:89所示的下游引物；针对如SEQ ID NO:30所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:90所示的上游引物和如SEQ ID NO:91所示的下游引物；针对如SEQ ID NO:31所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:92所示的上游引物和如SEQ ID NO:93所示的下游引物。Preferably, the PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:1 include: an upstream primer shown in SEQ ID NO:32 and a downstream primer shown in SEQ ID NO:33; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:2 include: upstream primers as shown in SEQ ID NO:34 and downstream primers as shown in SEQ ID NO:35; The PCR primers for amplification of the nucleotide sequence shown in: 3 include the upstream primer shown in SEQ ID NO: 36 and the downstream primer shown in SEQ ID NO: 37; for the nucleus shown in SEQ ID NO: 4 The PCR primers for nucleotide sequence amplification include upstream primers as shown in SEQ ID NO: 38 and downstream primers as shown in SEQ ID NO: 39; for the nucleotide sequence amplification as shown in SEQ ID NO: 5 PCR primers include upstream primers as shown in SEQ ID NO:40 and downstream primers as shown in SEQ ID NO:41; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:6 include as SEQ ID The upstream primer set forth in NO:42 and the downstream primer set forth in SEQ ID NO:43; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:7 include the set forth in SEQ ID NO:44 Upstream primers and downstream primers as set forth in SEQ ID NO:45; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:8 include upstream primers as set forth in SEQ ID NO:46 and as set forth in SEQ ID Downstream primer set forth in NO:47; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:9 include the upstream primer set forth in SEQ ID NO:48 and the upstream primer set forth in SEQ ID NO:49 Downstream primers; PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO: 10 include upstream primers as shown in SEQ ID NO: 50 and downstream primers as shown in SEQ ID NO: 51; for SEQ ID NO: 51 The PCR primers for amplification of the nucleotide sequence shown in ID NO: 11 include the upstream primer shown in SEQ ID NO: 52 and the downstream primer shown in SEQ ID NO: 53; The PCR primers for the amplification of the nucleotide sequence shown in NO: 12 include: the upstream primer shown in SEQ ID NO: 54 and the downstream primer shown in SEQ ID NO: 55; The PCR primers for the amplification of the nucleotide sequence include: upstream primers as shown in SEQ ID NO: 56 and downstream primers as shown in SEQ ID NO: 57; for those shown in SEQ ID NO: 14 PCR primers for nucleotide sequence amplification include upstream primers as shown in SEQ ID NO:58 and downstream primers as shown in SEQ ID NO:59; for amplification of nucleotide sequences as shown in SEQ ID NO:15 The PCR primers include the upstream primer as shown in SEQ ID NO:60 and the downstream primer as shown in SEQ ID NO:61; the PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:16 include as SEQ ID NO:16 The upstream primer set forth in ID NO:62 and the downstream primer set forth in SEQ ID NO:63; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:17 include set forth in SEQ ID NO:64 The upstream primers and the downstream primers as shown in SEQ ID NO:65; the PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:18 include the upstream primers as shown in SEQ ID NO:66 and as shown in SEQ ID NO:18 Downstream primer set forth in ID NO: 67; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO: 19 include the upstream primer set forth in SEQ ID NO: 68 and the upstream primer set forth in SEQ ID NO: 69 The downstream primers of ; PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:20 include the upstream primers shown in SEQ ID NO:70 and the downstream primers shown in SEQ ID NO:71; The PCR primers for the amplification of the nucleotide sequence shown in SEQ ID NO:21 include the upstream primer shown in SEQ ID NO:72 and the downstream primer shown in SEQ ID NO:73; The PCR primers for amplification of the nucleotide sequence shown include upstream primers as shown in SEQ ID NO: 74 and downstream primers as shown in SEQ ID NO: 75; The PCR primers for acid sequence amplification include: upstream primers as shown in SEQ ID NO: 76 and downstream primers as shown in SEQ ID NO: 77; for amplification of the nucleotide sequence as shown in SEQ ID NO: 24 PCR primers include: upstream primers as shown in SEQ ID NO: 78 and downstream primers as shown in SEQ ID NO: 79; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO: 25 include as SEQ ID NO: 25 The upstream primer set forth in ID NO: 80 and the downstream primer set forth in SEQ ID NO: 81; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO: 26 include those set forth in SEQ ID NO: 82 upstream primers and downstream primers as shown in SEQ ID NO: 83; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO: 27 include as in SEQ ID NO: 27 Upstream primers shown in D NO:84 and downstream primers shown in SEQ ID NO:85; PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:28 include those shown in SEQ ID NO:86 The upstream primer and the downstream primer as shown in SEQ ID NO:87; the PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:29 include the upstream primer as shown in SEQ ID NO:88 and as shown in SEQ ID NO:29 Downstream primer set forth in ID NO:89; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:30 include the upstream primer set forth in SEQ ID NO:90 and the upstream primer set forth in SEQ ID NO:91 The downstream primers of ; PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:31 include the upstream primers shown in SEQ ID NO:92 and the downstream primers shown in SEQ ID NO:93.
优选地，所述食源性致病菌包括以下菌种中的至少一种：单增李斯特菌、副溶血性弧菌、金黄色葡萄球菌、小肠结肠炎耶尔森氏菌、蜡样芽孢杆菌、克罗诺杆菌、致泻大肠埃希氏菌、沙门氏菌、空肠弯曲杆菌。Preferably, the food-borne pathogenic bacteria include at least one of the following bacteria species: Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Yersinia enterocolitica, Bacillus cereus Bacillus, Cronobacter, diarrhea-causing Escherichia coli, Salmonella, Campylobacter jejuni.
优选地，所述步骤1所述待测样品中的食源性致病菌菌体浓度控制在1×10 4～1×10 6cfu/mL范围内。 Preferably, the concentration of foodborne pathogenic bacteria in the sample to be tested in the step 1 is controlled within the range of 1×10 4 to 1×10 6 cfu/mL.
优选地，所述步骤1具体操作如下：在待测样品中加入PMA溶液，将其置于37℃恒温培养箱中避光培养5min，用500W钨丝灯照射10min。该步骤的目的是为了使死菌或细胞膜破损的菌的DNA与PMA交联，使其不能进行PCR扩增。Preferably, the specific operations of step 1 are as follows: add PMA solution to the sample to be tested, place it in a 37°C constant temperature incubator for 5 minutes in the dark, and irradiate it with a 500W tungsten lamp for 10 minutes. The purpose of this step is to cross-link the DNA of dead bacteria or bacteria with damaged cell membranes to PMA so that PCR amplification cannot be performed.
优选地，所述PMA终浓度为2～16mg/mL。更优选地，所述PMA终浓度为16mg/mL。本发明通过优化最终得出在该浓度条件下能最同时抑制上述食源性致病死菌的DNA。Preferably, the final concentration of PMA is 2-16 mg/mL. More preferably, the final concentration of PMA is 16 mg/mL. The present invention finally obtains the DNA that can inhibit the above-mentioned food-borne pathogenic and dead bacteria at the same time under the condition of this concentration through optimization.
优选地，所述步骤2提取菌体DNA采用磁珠法提取菌悬液中的DNA。Preferably, in the step 2, the bacterial cell DNA is extracted by magnetic bead method to extract the DNA in the bacterial suspension.
优选地，所述步骤3中实时荧光定量PCR的反应体系为：2×qPCR反应缓冲液10μL，5μM上游引物0.4μL，5μM下游引物0.4μL，DNA模板2μL，灭菌双蒸水7.2μL，总体积20μL。Preferably, the reaction system of real-time fluorescence quantitative PCR in the step 3 is: 10 μL of 2×qPCR reaction buffer, 0.4 μL of 5 μM upstream primer, 0.4 μL of 5 μM downstream primer, 2 μL of DNA template, 7.2 μL of sterilized double-distilled water, and the total Volume 20 μL.
优选地，所述步骤3中实时荧光定量PCR的反应程序为：(1)预变性：95℃，30s，20℃/s，1Cycle；(2)PCR扩增反应：95℃，5s，20℃/s，60℃，5s，20℃/s，40Cycles；(3)溶解曲线分析：95℃，0s，20℃/s，65℃，15s，20℃/s，95℃，0s，0.1℃/s。Preferably, the reaction procedure of real-time fluorescence quantitative PCR in the step 3 is: (1) pre-denaturation: 95°C, 30s, 20°C/s, 1Cycle; (2) PCR amplification reaction: 95°C, 5s, 20°C /s, 60℃, 5s, 20℃/s, 40Cycles; (3) Dissolution curve analysis: 95℃, 0s, 20℃/s, 65℃, 15s, 20℃/s, 95℃, 0s, 0.1℃/ s.
本发明的有益效果为：本发明公开了用于识别食源性致病菌所携带的40个特异性基因靶点及相关PMA-qPCR定量检测方法；与现有技术相比，本发明的检测方法具有检测时间短，无须经过传统的培养增菌过程，不受样品中食源性致病菌标准菌株死菌干扰，准确检测并定量样品中食源性致病菌标准菌株的具体含菌数量，有效避免假阳性结果。并且选取的分子靶标为各个食源性标准菌株的独有核酸序列，检测结果特异性强，与其它近缘、同缘菌株无交叉反应，结果判定简单，检测结果与传统平板计数方法基本一致。The beneficial effects of the present invention are as follows: the present invention discloses 40 specific gene targets and related PMA-qPCR quantitative detection methods for identifying food-borne pathogenic bacteria; The method has the advantages of short detection time, does not need to go through the traditional culture and enrichment process, and is not interfered by the dead bacteria of the standard food-borne pathogenic bacteria in the sample, and can accurately detect and quantify the specific bacteria quantity of the standard food-borne pathogenic bacteria in the sample. , to effectively avoid false positive results. In addition, the selected molecular targets are the unique nucleic acid sequences of each food-borne standard strain. The detection results are highly specific and have no cross-reaction with other closely related and homologous strains. The results are simple to determine, and the detection results are basically consistent with the traditional plate counting method.
附图说明Description of drawings
图1为PMA抑制死菌DNA扩增效果评价qPCR结果及不同浓度PMA处理标准菌株电泳图：(1)2-16分别为浓度2-16μg/mLPMA处理后跑胶结果，M为2000Maker，(2)PMA处理和未处理的含有死菌和含有活菌样品qPCR结果图；Figure 1 shows the qPCR results for the evaluation of the effect of PMA on the DNA amplification of dead bacteria and the electrophoresis of standard strains treated with different concentrations of PMA: (1) 2-16 are the results of gel running after treatment with concentrations of 2-16 μg/mL PMA, M is 2000Maker, (2 ) qPCR results of PMA-treated and untreated samples containing dead bacteria and samples containing viable bacteria;
图2为PMA处理和未处理的含有不同浓度梯度食源性致病菌样品的PCR电泳结果图；Figure 2 shows the results of PCR electrophoresis of PMA-treated and untreated food-borne pathogenic bacteria samples with different concentration gradients;
图3为PMA处理方法对各食源性致病菌死菌抑制效果；Fig. 3 is the inhibitory effect of PMA treatment method on the dead bacteria of various food-borne pathogens;
图4为食源性大肠杆菌活细胞检测标准曲线；Figure 4 is a standard curve for the detection of foodborne Escherichia coli live cells;
图5为食源性沙门氏菌活细胞检测标准曲线；Fig. 5 is the standard curve of live cell detection of foodborne Salmonella;
图6为食源性克罗诺杆菌活细胞检测标准曲线；Figure 6 is the standard curve of foodborne Cronobacter live cell detection;
图7为食源性副溶血性弧菌活细胞检测标准曲线；Figure 7 is a standard curve for the detection of food-borne Vibrio parahaemolyticus viable cells;
图8为食源性单增李斯特菌活细胞检测标准曲线；Fig. 8 is the standard curve of live cell detection of foodborne Listeria monocytogenes;
图9为食源性蜡样芽胞杆菌活细胞检测标准曲线；Fig. 9 is the standard curve of live cell detection of foodborne Bacillus cereus;
图10为食源性小肠结肠炎耶尔森氏菌活细胞检测标准曲线；Figure 10 is a standard curve for the detection of foodborne Yersinia enterocolitica live cells;
图11为食源性空肠弯曲菌活细胞检测标准曲线；Figure 11 is the standard curve for the detection of food-borne Campylobacter jejuni live cells;
图12为食源性金黄色葡萄球菌活细胞检测标准曲线。Figure 12 is the standard curve for the detection of food-borne Staphylococcus aureus live cells.
具体实施方式Detailed ways
为了更加简洁明了的展示本发明的技术方案、目的和优点，下面结合具体实施例和附图详细说明本发明的技术方案。In order to show the technical solutions, objects and advantages of the present invention more concisely and clearly, the technical solutions of the present invention are described in detail below with reference to specific embodiments and accompanying drawings.
实施例1Example 1
一、PMA-qPCR检测方法的建立：1. Establishment of PMA-qPCR detection method:
1、检测引物的设计1. Design of detection primers
通过比较基因组学分析，进行同源比对分析，获得以下食源性致病菌的特异性分子靶标，然后针对这些特异性分子靶标设计引物(引物由上海生物工程技术服务有限公司合成)，以便进行PCR扩增，具体信息如表1所示：Through comparative genomics analysis, homologous alignment analysis was performed to obtain the specific molecular targets of the following food-borne pathogens, and then primers were designed for these specific molecular targets (the primers were synthesized by Shanghai Bioengineering Technology Service Co., Ltd.), so that PCR amplification was performed, and the specific information is shown in Table 1:
表1：各个食源性致病菌的特异性分子靶标及其引物Table 1: Specific molecular targets and primers for each foodborne pathogen
Figure PCTCN2021087080-appb-000001
Figure PCTCN2021087080-appb-000001
Figure PCTCN2021087080-appb-000002
Figure PCTCN2021087080-appb-000002
Figure PCTCN2021087080-appb-000003
Figure PCTCN2021087080-appb-000003
二、PMA-qPCR检测体系构建：2. Construction of PMA-qPCR detection system:
步骤1：在待测样品加入PMA染料溶液，具体操作如下；Step 1: Add PMA dye solution to the sample to be tested, the specific operation is as follows;
取1mL样品于EP管中，在10000r/min条件下离心5min弃上清液，向沉淀中加入1mL去离子水复溶，加入终浓度为16mg/mL的PMA后采用震荡混合仪涡旋至沉淀悬浮且分散均匀，将其以铝箔纸包裹置于37℃恒温培养箱中避光培养5min，避光培养完成后，用500W钨丝灯照射10min使PMA与DNA充分交联，10000r/min离心5min得到沉淀，用1mL去离子水复溶，再次离心，洗涤3次去除体系中多余的PMA。Take 1 mL of the sample into an EP tube, centrifuge at 10,000 r/min for 5 min to discard the supernatant, add 1 mL of deionized water to the precipitate to reconstitute, add PMA with a final concentration of 16 mg/mL, and then vortex with a vibrating mixer to precipitate Suspended and uniformly dispersed, wrapped in aluminum foil and placed in a constant temperature incubator at 37 °C for 5 min in the dark. After the dark incubation, irradiated with a 500W tungsten filament lamp for 10 min to fully cross-link PMA and DNA, and centrifuged at 10,000 r/min for 5 min. The precipitate was obtained, reconstituted with 1 mL of deionized water, centrifuged again, and washed 3 times to remove excess PMA in the system.
步骤2：提取待测样品中的菌体DNA，具体操作如下；Step 2: Extract the bacterial DNA in the sample to be tested, and the specific operations are as follows;
采用磁珠法提取菌悬液NDA，将取处理后的菌悬液10000r/min下离心，尽可能除尽上清液，于沉淀中加入包含溶菌酶的180μLTE buffer，涡旋震荡使体系充分混合，37℃水浴(革兰氏阳性菌60min，革兰氏阴性菌30min)。加入500μL裂解液和20μL蛋白酶K，剧烈震荡15s以充分混匀，56℃温浴20min，离心管加入15μL磁珠并颠倒混匀，通过磁性分离架吸附磁珠去除上清加入500μL去蛋白漂洗液，再次反复颠倒离心管数次确保磁珠完全分散，再次使用磁性分离架吸附磁珠加入800μL洗涤液清洗，磁性分离后开盖干燥5min，最终加入100μL洗脱液在70℃下水浴5分钟，获得目标DNA溶液。The bacterial suspension NDA was extracted by the magnetic bead method, and the treated bacterial suspension was centrifuged at 10,000 r/min, the supernatant was removed as much as possible, and 180 μL LTE buffer containing lysozyme was added to the precipitate, and the system was fully mixed by vortexing. , 37 ℃ water bath (60min for Gram-positive bacteria, 30min for Gram-negative bacteria). Add 500 μL of lysis solution and 20 μL of proteinase K, shake vigorously for 15 s to fully mix, incubate at 56 °C for 20 min, add 15 μL of magnetic beads to the centrifuge tube and mix by inversion, remove the supernatant by adsorbing the magnetic beads on a magnetic separation rack, and add 500 μL of deproteinized rinse solution. Invert the centrifuge tube several times to ensure that the magnetic beads are completely dispersed. Use the magnetic separation rack to absorb the magnetic beads again and add 800 μL of washing solution to wash. After magnetic separation, open the lid and dry for 5 minutes. Finally, add 100 μL of the eluent to a water bath at 70 °C for 5 minutes to obtain target DNA solution.
步骤3：以步骤2中的菌体DNA为模板，进行实时荧光定量PCR检测，具体操作如下；Step 3: Use the bacterial DNA in Step 2 as a template to perform real-time fluorescence quantitative PCR detection, and the specific operations are as follows;
根据上述表1针对各个特异性引物设计的引物和荧光染料嵌入法(q-PCR)对各个食源性致病菌的特异性核酸序列进行定量检测，扩增参数具体为：The specific nucleic acid sequences of each food-borne pathogenic bacteria are quantitatively detected according to the primers designed for each specific primer and the fluorescent dye intercalation method (q-PCR) according to the above-mentioned table 1, and the amplification parameters are specifically:
qPCR反应体系为：The qPCR reaction system is:
Figure PCTCN2021087080-appb-000004
Figure PCTCN2021087080-appb-000004
qPCR反应程序为:The qPCR reaction program is:
(1)预变性(1) Pre-denaturation
95℃   30s   20℃/s95℃ 30s 20℃/s
1Cycle；1Cycle;
(2)PCR反应(2) PCR reaction
95℃   5s   20℃/s95℃ 5s 20℃/s
60℃   5s   20℃/s60℃ 5s 20℃/s
40Cycles；40Cycles;
(3)溶解曲线分析(3) Dissolution curve analysis
95℃   0s   20℃/s95℃ 0s 20℃/s
65℃   15s  20℃/s65℃ 15s 20℃/s
95℃   0s   0.1℃/s。95℃ 0s 0.1℃/s.
步骤4：待上述反应结束后，将获得的荧光定量PCR扩增的曲线和Ct值与建立的标准曲线计算样品中食源性致病菌活菌的数量；具体操作如下：Step 4: After the above reaction is completed, calculate the number of viable food-borne pathogenic bacteria in the sample by comparing the obtained curve and Ct value of fluorescence quantitative PCR amplification with the established standard curve; the specific operations are as follows:
各个食源性致病菌标准曲线的建立：取各个表1中的各个菌的高浓度活菌悬液首先按10倍梯度连续稀释8次，通过平板计数法确定菌液浓度，将菌液取出三份，每份1ml，照上述步骤提取DNA。按照q-PCR步骤进行扩增， 获得荧光曲线图和有效的Ct值(Ct＜35)，并根据origin计算得到标准曲线如图4～12所示：The establishment of the standard curve of each foodborne pathogen Three aliquots of 1 ml each were used to extract DNA as described above. Amplify according to the q-PCR step to obtain the fluorescence curve and effective Ct value (Ct<35), and calculate the standard curve according to the origin, as shown in Figures 4-12:
(1)致泻大肠埃希氏菌活细胞检测标准曲线如图4所示：(1) The standard curve of live cell detection of diarrhea-causing Escherichia coli is shown in Figure 4:
菌株PY002的R2为0.97924，检测极限为10 2cfu/mL；菌株2968A1的R2为0.99747，检测极限为10 2cfu/mL；菌株3164A1的R2为0.97441，检测极限为10 3cfu/mL；菌株3466A3的R2为0.98487，检测极限为10 2cfu/mL；菌株3025B1的R2为0.98813，检测极限为10 2cfu/mL；菌株3776A3-1的R2为0.99487，检测极限为10 2cfu/mL。 Strain PY002 has an R2 of 0.97924 with a detection limit of 10 2 cfu/mL; strain 2968A1 has an R2 of 0.99747 with a detection limit of 10 2 cfu/mL; strain 3164A1 has an R2 of 0.97441 with a detection limit of 10 3 cfu/mL; strain 3466A3 The R2 of strain 3025B1 was 0.98487, and the detection limit was 10 2 cfu/mL; the R2 of strain 3025B1 was 0.98813, and the detection limit was 10 2 cfu/mL; the R2 of strain 3776A3-1 was 0.99487, and the detection limit was 10 2 cfu/mL.
(2)致泻沙门氏菌活细胞检测标准曲线如图5：(2) The standard curve of live cell detection of Salmonella diarrhoea is shown in Figure 5:
菌株FSCC(I)215032的R2为0.98151，检测极限为10 2cfu/mL；菌株FSCC(I)21501206的R2为0.9767，检测极限为10 2cfu/mL；菌株FSCC(I)215467的R2为0.99947，检测极限为10 2cfu/mL；菌株FSCC(I)215456的R2为0.98519，检测极限为10 2cfu/mL；菌株FSCC(I)215032的R2为0.99634，检测极限为10 2cfu/mL。 The R2 of strain FSCC(I)215032 was 0.98151, and the detection limit was 10 2 cfu/mL; the R2 of strain FSCC(I)21501206 was 0.9767, and the detection limit was 10 2 cfu/mL; the R2 of strain FSCC(I) 215467 was 0.99947 , the detection limit was 10 2 cfu/mL; the R2 of strain FSCC(I)215456 was 0.98519, and the detection limit was 10 2 cfu/mL; the R2 of strain FSCC(I) 215032 was 0.99634, and the detection limit was 10 2 cfu/mL.
(3)克罗诺杆菌标准菌株活细胞检测标准曲线如图6：(3) The standard curve of Cronobacter standard strain live cell detection is shown in Figure 6:
菌株cro359W的R2为0.9964，极限为10 2cfu/mL；菌株cro509C1的R2为0.99483，检测极限为10 3cfu/mL；菌株cro611A3的R2为0.98703，检测极限为10 2cfu/mL；菌株cro910B3的R2为0.997，检测极限为10 4cfu/mL；菌株cro1537W的R2为0.99424，检测极限为10 3cfu/mL。 The R2 of strain cro359W is 0.9964, and the limit is 10 2 cfu/mL; the R2 of strain cro509C1 is 0.99483, and the detection limit is 10 3 cfu/mL; the R2 of strain cro611A3 is 0.98703, and the detection limit is 10 2 cfu/mL; The R2 was 0.997, and the detection limit was 10 4 cfu/mL; the R2 of the strain cro1537W was 0.99424, and the detection limit was 10 3 cfu/mL.
(3)副溶血性弧菌活细胞检测标准曲线如图7：(3) The standard curve of Vibrio parahaemolyticus live cell detection is shown in Figure 7:
菌株VP2227C2的R2为0.99359，检测极限为10 3cfu/mL；菌株VPS179C3的R2为0.98814，检测极限为10 3cfu/mL；菌株3630A3的R2为0.96913，检测极限为10 2cfu/mL。 The R2 of strain VP2227C2 was 0.99359, and the detection limit was 10 3 cfu/mL; the R2 of strain VPS179C3 was 0.98814, and the detection limit was 10 3 cfu/mL; the R2 of strain 3630A3 was 0.96913, and the detection limit was 10 2 cfu/mL.
(5)单增李斯特菌活细胞检测标准曲线如图8：(5) The standard curve of live cell detection of Listeria monocytogenes is shown in Figure 8:
菌株428-1LM的R2为0.99454，检测极限为10 3cfu/mL；菌株615-1LM的R2为0.9695，检测极限为10 2cfu/mL；标准菌株678-1LM的R2为0.98931，检测极限为10 4cfu/mL；菌株833-1LM的R2为0.99363，检测极限为10 3cfu/mL；菌株1382-1LM的R2为0.95412，检测极限为10 3cfu/mL。 Strain 428-1LM has an R2 of 0.99454 with a detection limit of 10 3 cfu/mL; strain 615-1LM has an R2 of 0.9695 with a detection limit of 10 2 cfu/mL; standard strain 678-1LM has an R2 of 0.98931 with a detection limit of 10 4 cfu/mL; strain 833-1LM had an R2 of 0.99363 with a detection limit of 10 3 cfu/mL; strain 1382-1LM had an R2 of 0.95412 with a detection limit of 10 3 cfu/mL.
(6)蜡样芽胞杆菌标准菌株活细胞检测标准曲线如图9：(6) The standard curve of Bacillus cereus standard strain live cell detection is shown in Figure 9:
菌株260-1B的R2为0.97443，检测极限为10 3cfu/mL；菌株Y1712的R2为0.98046，检测极限为10 3cfu/mL；菌株1761-2A的R2为0.99759，检测极限为10 3cfu/mL；菌株2801的R2为0.99613，检测极限为10 2cfu/mL； The R2 of strain 260-1B was 0.97443, and the detection limit was 10 3 cfu/mL; the R2 of strain Y1712 was 0.98046, and the detection limit was 10 3 cfu/mL; the R2 of strain 1761-2A was 0.99759, and the detection limit was 10 3 cfu/mL. mL; the R2 of strain 2801 was 0.99613, and the detection limit was 10 2 cfu/mL;
菌株2841-1B的R2为0.99367，检测极限为10 3cfu/mL。 The R2 of strain 2841-1B was 0.99367 with a detection limit of 103 cfu/mL.
(7)小肠结肠炎耶尔森氏菌活细胞检测标准曲线如图10：(7) The standard curve of Yersinia enterocolitica live cell detection is shown in Figure 10:
菌株c009的R2为0.99942，检测极限为10 2cfu/mL；菌株y802的R2为0.99615，检测极限为10 2cfu/mL；菌株c1702的R2为0.9906，检测极限为10 1cfu/mL。 The R2 of strain c009 was 0.99942, and the detection limit was 10 2 cfu/mL; the R2 of strain y802 was 0.99615, and the detection limit was 10 2 cfu/mL; the R2 of strain c1702 was 0.9906, and the detection limit was 10 1 cfu/mL.
(8)空肠弯曲菌活细胞检测标准曲线如图11：(8) The standard curve of live cell detection of Campylobacter jejuni is shown in Figure 11:
菌株GDMCC 60857的R2为0.96844，检测极限为10 4cfu/mL；菌株GDMCC 60858的R2为0.96895，检测极限为10 5cfu/mL。 The R2 of strain GDMCC 60857 was 0.96844 with a detection limit of 10 4 cfu/mL; the R2 of strain GDMCC 60858 was 0.96895 with a detection limit of 10 5 cfu/mL.
(9)金黄色葡萄球菌活细胞检测标准曲线如图12：(9) The standard curve of Staphylococcus aureus live cell detection is shown in Figure 12:
菌株Sta144-2的R2为0.96381，检测极限为10 2cfu/mL；菌株Sta403的R2为0.99793，检测极限为10 2cfu/mL；菌株Sta177-0的R2为0.99796，检测极限为10 5cfu/mL；菌株Sta1942-0的R2为0.99473，检测极限为10 5cfu/mL；菌株Sta370B3的R2为0.99942，检测极限为10 3cfu/mL；菌株Sta4127的R2为0.983，检测极限为10 4cfu/mL。 The R2 of strain Sta144-2 is 0.96381, and the detection limit is 10 2 cfu/mL; the R2 of strain Sta403 is 0.99793, and the detection limit is 10 2 cfu/mL; the R2 of strain Sta177-0 is 0.99796, and the detection limit is 10 5 cfu/mL mL; the R2 of strain Sta1942-0 was 0.99473, and the detection limit was 10 5 cfu/mL; the R2 of strain Sta370B3 was 0.99942, and the detection limit was 10 3 cfu/mL; the R2 of strain Sta4127 was 0.983, and the detection limit was 10 4 cfu/mL mL.
实施例2 PMA-qPCR检测体系PMA条件优化 Embodiment 2 PMA-qPCR detection system PMA condition optimization
1、死菌未进行PMA处理的对比试验1. Comparative test of dead bacteria without PMA treatment
以沙门氏菌FSCC(I)215467为例，取10 6cfu/mL数量级菌液4mL分成4份，2份通过加热制备为死菌样品。取一份活菌样品和死菌样品采用PMA处理：离心去上清液，加入1mL去离子水复溶，再加入1mg/mL的PMA溶液10μL，漩涡震荡10s，盖上铝箔纸摇床室温避光孵育5min，采用500W钨丝灯照射10分钟，使PMA与死菌DNA充分交联。10000r/min条件下离心5分钟，去上清，再次用去1mL离子水复溶，漩涡震荡10s，再次离心，重复以上步骤3次，彻底去除未结合的PMA成分。4份样品按照提取DNA流程获得其DNA溶液，分别标记为Killed-PMA(死菌未经PMA处理)，Lived-PMA(活菌未经PMA处理)，Lived+PMA(活菌PMA处理)，Killed+PMA(死菌PMA处理)，进行qPCR扩增检测。结果如图1(1)所示，Lived-PMA和Killed-PMA的Ct值最小且数值接近，其次是Lived+PMA，Killed+PMA的Ct值最小，说明PMA处理在具有显著抑制死菌扩增的效果。 Taking Salmonella FSCC(I) 215467 as an example, 4 mL of 10 6 cfu/mL bacterial solution was taken and divided into 4 parts, and 2 parts were prepared as dead bacteria samples by heating. Take a sample of live bacteria and dead bacteria and treat with PMA: centrifuge to remove the supernatant, add 1 mL of deionized water to reconstitute, then add 10 μL of 1 mg/mL PMA solution, vortex for 10 s, cover with aluminum foil and shake at room temperature. Incubate with light for 5 min, and irradiate with a 500W tungsten filament lamp for 10 min to fully cross-link PMA with dead bacterial DNA. Centrifuge at 10,000 r/min for 5 minutes, remove the supernatant, reconstitute with 1 mL of ionized water, vortex for 10 s, centrifuge again, and repeat the above steps 3 times to completely remove unbound PMA components. The DNA solutions of 4 samples were obtained according to the DNA extraction process, which were marked as Killed-PMA (dead bacteria without PMA treatment), Lived-PMA (live bacteria without PMA treatment), Lived+PMA (live bacteria without PMA treatment), Killed +PMA (dead bacteria PMA treatment) for qPCR amplification detection. The results are shown in Figure 1(1), the Ct values of Lived-PMA and Killed-PMA are the smallest and close to each other, followed by Lived+PMA, and the Ct value of Killed+PMA is the smallest, indicating that PMA treatment can significantly inhibit the amplification of dead bacteria. Effect.
2、PMA终浓度对死菌DNA扩增的影响2. The effect of the final concentration of PMA on the DNA amplification of dead bacteria
取10 6cfu/mL数量级沙门氏菌FSCC(I)215467菌液8mL经过加热制成死菌悬浮液后涡旋等分封装至8个1mL离心管，在10000r/min条件下离心5min，用去离子水复溶至1mL，并分别使得PMA终浓度为2、4、6、8、10、12、14、16μg/mL，并通过其特异性引物的PCR扩增，电泳跑胶观察PCR产物，如图1(2)，跑胶条带荧光强度随着PMA量的增加呈现递减趋势，至16μg/mL时可以完全抑制死菌DNA的核酸扩增。 Take 8 mL of 10 6 cfu/mL Salmonella FSCC(I) 215467 bacterial solution, heat it to make dead bacteria suspension, vortex aliquot and pack it into 8 1 mL centrifuge tubes, centrifuge at 10000 r/min for 5 min, use deionized water Reconstituted to 1 mL, and the final concentration of PMA was 2, 4, 6, 8, 10, 12, 14, 16 μg/mL, respectively, and amplified by PCR with its specific primers, and the PCR products were observed by electrophoresis, as shown in the figure. 1(2), the fluorescence intensity of the running strip shows a decreasing trend with the increase of the amount of PMA, and when it reaches 16 μg/mL, the nucleic acid amplification of dead bacterial DNA can be completely inhibited.
3、菌体浓度对PMA处理结果的影响3. The effect of bacterial concentration on the results of PMA treatment
以沙门氏菌株FSCC(I)215032,FSCC(I)21501206,FSCC(I)215467为考察对象，通过梯度稀释分别制备含有不同浓度标准菌株含量(10 1-10 8cfu/mL)的样品。经过PMA处理和未处理的样品电泳跑胶结果如图3所示，在相同浓度条件下，经过PMA处理的样品条带荧光强度明显弱于左侧未经PMA处理样品，对FSCC(I)215032的检测灵敏度从10 5降至10 6cfu/mL,对FSCC(I)21501206的检测灵敏度从10 3降至10 5cfu/mL,对FSCC(I)215467的检测灵敏度从10 6降至10 7cfu/mL。说明在处理实际样品中PMA-PCR法可以有效去除常见的浓度梯度中死菌DNA的干扰，减少假阳性结果。 Taking Salmonella strains FSCC(I) 215032, FSCC(I) 21501206 and FSCC(I) 215467 as investigation objects, samples containing different concentrations of standard strains (10 1 -10 8 cfu/mL) were prepared by gradient dilution. The electrophoresis results of the samples treated with PMA and untreated are shown in Figure 3. Under the same concentration conditions, the fluorescence intensity of the samples treated with PMA was significantly weaker than that of the samples without PMA treatment on the left. The detection sensitivity decreased from 10 5 to 10 6 cfu/mL, the detection sensitivity of FSCC(I)21501206 decreased from 10 3 to 10 5 cfu/mL, and the detection sensitivity of FSCC(I)215467 decreased from 10 6 to 10 7 cfu/mL. It shows that PMA-PCR method can effectively remove the interference of dead bacterial DNA in common concentration gradients and reduce false positive results in the processing of actual samples.
4、PMA-PCR方法对各食源性致病菌抑制数量4. PMA-PCR method inhibits the number of foodborne pathogens
PMA加入量过多会造成试剂的浪费和假阴性结果，加入量过少可能不能完全抑制死菌浓度。制备各个食源性致病菌常见浓度的死菌悬浮液检测PMA-PCR方法抑制各株死菌浓度最高极限，检测结果如图4所示。16μg/mLPMA浓度可以有效抑制各个标准菌株的最高浓度为：副溶血性弧菌(10 6cfu/mL)、致泻大肠埃希氏菌(10 6cfu/mL)、单增李斯特菌(10 6cfu/mL)、金黄色葡萄球菌(10 5cfu/mL)、克罗诺杆菌(10 5cfu/mL)、蜡样芽胞杆菌(10 4cfu/mL)、沙门氏菌(10 6cfu/mL)、小肠结肠炎耶尔森氏菌(10 6cfu/mL)、空肠弯曲菌(10 4cfu/mL)。 Adding too much PMA will cause waste of reagents and false negative results, and adding too little may not completely suppress the dead bacteria concentration. The dead bacteria suspension with the common concentration of each foodborne pathogen was prepared to detect the highest limit of the dead bacteria concentration of each strain by PMA-PCR method. The test results are shown in Figure 4. The highest concentration of 16μg/mL PMA that can effectively inhibit each standard strain is: Vibrio parahaemolyticus (10 6 cfu/mL), diarrhea-causing Escherichia coli (10 6 cfu/mL), Listeria monocytogenes (10 6 cfu/mL), Staphylococcus aureus (10 5 cfu/mL), Cronobacter (10 5 cfu/mL), Bacillus cereus (10 4 cfu/mL), Salmonella (10 6 cfu/mL) , Yersinia enterocolitica (10 6 cfu/mL), Campylobacter jejuni (10 4 cfu/mL).
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。The above-mentioned embodiments only represent several embodiments of the present invention, and the descriptions thereof are specific and detailed, but should not be construed as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can also be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (10)

  1. 用于检测食源性致病菌的特异性分子靶标，其特征在于，所述特异性分子靶标的核苷酸序列如SEQ ID NO:1～31所示。A specific molecular target for detecting food-borne pathogens, characterized in that the nucleotide sequence of the specific molecular target is shown in SEQ ID NOs: 1-31.
  2. 如权利要求1所述的特异性分子靶标，其特征在于，所述特异性分子靶标使用核酸序列如SEQ ID NO：32～93所示的引物分别检测。The specific molecular target according to claim 1, wherein the specific molecular target is detected using primers whose nucleic acid sequences are shown in SEQ ID NOs: 32-93, respectively.
  3. 如权利要求1所述的特异性分子靶标，其特征在于，所述食源性致病菌包括单增李斯特菌、副溶血性弧菌、金黄色葡萄球菌、小肠结肠炎耶尔森氏菌、蜡样芽孢杆菌、克罗诺杆菌、致泻大肠埃希氏菌、沙门氏菌和空肠弯曲杆菌。The specific molecular target of claim 1, wherein the food-borne pathogenic bacteria include Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, and Yersinia enterocolitica , Bacillus cereus, Cronobacter, diarrhea-causing Escherichia coli, Salmonella, and Campylobacter jejuni.
  4. 一种食源性致病菌活菌的检测方法，其特征在于，包括以下步骤：A kind of detection method of food-borne pathogenic bacteria viable bacteria, is characterized in that, comprises the following steps:
    步骤1：在待测样品加入PMA染料溶液；Step 1: Add PMA dye solution to the sample to be tested;
    步骤2：提取待测样品中的菌体DNA；Step 2: Extract the bacterial DNA in the sample to be tested;
    步骤3：以步骤2中的菌体DNA为模板，进行实时荧光定量PCR检测；Step 3: use the bacterial DNA in step 2 as a template to perform real-time fluorescence quantitative PCR detection;
    步骤4：待反应结束后，将获得的荧光定量PCR扩增的曲线和Ct值与建立的标准曲线计算样品中食源性致病菌活菌的数量；Step 4: After the reaction is completed, calculate the number of viable food-borne pathogenic bacteria in the sample by comparing the obtained curve and Ct value of fluorescence quantitative PCR amplification with the established standard curve;
    其中，所述步骤3中进行实时荧光定量PCR检测所使用的引物包括如SEQ ID NO：32～93所示的核酸序列。Wherein, the primers used for real-time fluorescence quantitative PCR detection in the step 3 include nucleic acid sequences as shown in SEQ ID NOs: 32-93.
  5. 如权利要求4所述的方法，其特征在于，所述步骤2中实时荧光定量PCR检测扩增的目的片段的核苷酸序列如SEQ ID NO：1～31所示。The method of claim 4, wherein the nucleotide sequence of the target fragment amplified by real-time fluorescence quantitative PCR in the step 2 is shown in SEQ ID NOs: 1-31.
  6. 如权利要求5所述的方法，其特征在于，所述针对如SEQ ID NO:1所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:32所示的上游引物和如SEQ ID NO:33所示的下游引物；针对如SEQ ID NO:2所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:34所示的上游引物和如SEQ ID NO:35所示的下游引物；针对如SEQ ID NO:3所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:36所示的上游引物和如SEQ ID NO:37所示的下游引物；针对如SEQ ID NO:4所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:38所示的上游引物和如SEQ ID NO:39所示的下游引物；针对如SEQ ID NO:5所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:40所示的上游引物和如SEQ ID NO:41所示的下游引物；针对如SEQ ID NO:6所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:42所示的上游引物和如SEQ ID NO:43所示的下游引物；针对如SEQ ID NO:7所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:44所示的上游引物和如SEQ ID NO:45所示的下游引物；针对如SEQ ID NO:8所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:46所示的上游引物和如SEQ ID NO:47所示的下游引物；针对如SEQ ID NO:9所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:48所示的上游引物和如SEQ ID NO:49所示的下游引物；针对如SEQ ID NO:10所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:50所示的上游引物和如SEQ ID NO:51所示的下游引物；针对如SEQ ID NO:11所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:52所示的上游引物和如SEQ ID NO:53所示的下游引物；优选地，所述针对如SEQ ID NO:12所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:54所示的上游引物和如SEQ ID NO:55所示的下游引物；针对如SEQ ID NO:13所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:56所示的上游引物和如SEQ ID NO:57所示的下游引物；针对如SEQ ID NO:14所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:58所示的上游引物和如SEQ ID NO:59所示的下游引物；针对如SEQ ID NO:15所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:60所示的上游引物和如SEQ ID NO:61所示的下游引物；针对如SEQ ID NO:16所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:62所示的上游引物和如SEQ ID NO:63所示的下游引物；针对如SEQ ID NO:17所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:64所示的上游引物和如SEQ ID NO:65所示的下游引物；针对如SEQ ID NO:18所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:66所示的上游引物和如SEQ ID NO:67所示的下游引物；针对如SEQ ID NO:19所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:68所示的上游引物和如SEQ ID NO:69所示的下游引物；针对如SEQ ID NO:20所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:70所示的上游引物和如SEQ ID NO:71所示的下游引物；针对如SEQ ID NO:21所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:72所示的上游引物和如SEQ ID NO:73所示的下游引物；针对如SEQ ID NO:22所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:74所示的上游引物和如SEQ ID NO:75所示的下游引物；所述针对如SEQ ID NO:23所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:76所示的上游引物和如SEQ ID NO:77所示的下游引物；针对如SEQ ID NO:24所示的核苷酸序列扩增的PCR引物包括：如SEQ ID NO:78所示的上游引物和如SEQ ID NO:79所示的下游引物；针对如SEQ ID NO:25所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:80所示的上游引物和如SEQ ID NO:81所示的下游引物；针对如SEQ ID NO:26所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:82所示的上游引物和如SEQ ID NO:83所示的下游引物；针对如SEQ ID NO:27所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:84所示的上游引物和如SEQ ID NO:85所示的下游引物；针对如SEQ ID NO:28所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:86所示的上游引物和如SEQ ID NO:87所示的下游引物；针对如SEQ ID NO:29所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:88 所示的上游引物和如SEQ ID NO:89所示的下游引物；针对如SEQ ID NO:30所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:90所示的上游引物和如SEQ ID NO:91所示的下游引物；针对如SEQ ID NO:31所示的核苷酸序列扩增的PCR引物包括如SEQ ID NO:92所示的上游引物和如SEQ ID NO:93所示的下游引物。The method of claim 5, wherein the PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO: 1 include: an upstream primer shown in SEQ ID NO: 32 and an upstream primer shown in SEQ ID NO: 32 Downstream primer set forth in ID NO:33; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:2 include: upstream primer set forth in SEQ ID NO:34 and upstream primer set forth in SEQ ID NO:35 The downstream primers shown; PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:3 include the upstream primers shown in SEQ ID NO:36 and the downstream primers shown in SEQ ID NO:37; for PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:4 include the upstream primer set forth in SEQ ID NO:38 and the downstream primer set forth in SEQ ID NO:39; for SEQ ID NO:5 PCR primers for amplification of the nucleotide sequences shown include the upstream primer shown in SEQ ID NO:40 and the downstream primer shown in SEQ ID NO:41; for the nucleotide shown in SEQ ID NO:6 PCR primers for sequence amplification include upstream primers as shown in SEQ ID NO:42 and downstream primers as shown in SEQ ID NO:43; PCR primers for amplification of nucleotide sequences as shown in SEQ ID NO:7 Include an upstream primer as shown in SEQ ID NO:44 and a downstream primer as shown in SEQ ID NO:45; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:8 include as in SEQ ID NO: The upstream primer shown in 46 and the downstream primer shown in SEQ ID NO:47; PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:9 include the upstream primer shown in SEQ ID NO:48 and downstream primers as shown in SEQ ID NO:49; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:10 include upstream primers as shown in SEQ ID NO:50 and as shown in SEQ ID NO: Downstream primers shown in 51; PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:11 include upstream primers shown in SEQ ID NO:52 and downstream primers shown in SEQ ID NO:53 Preferably, the PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO: 12 include: upstream primers as shown in SEQ ID NO: 54 and downstream primers as shown in SEQ ID NO: 55 ; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO: 13 include: upstream primers as shown in SEQ ID NO: 56 and downstream primers as shown in SEQ ID NO: 57; for S The PCR primers for the amplification of the nucleotide sequence shown in EQ ID NO: 14 include the upstream primer shown in SEQ ID NO: 58 and the downstream primer shown in SEQ ID NO: 59; PCR primers for amplification of the nucleotide sequence shown include the upstream primer shown in SEQ ID NO:60 and the downstream primer shown in SEQ ID NO:61; for the nucleotide sequence shown in SEQ ID NO:16 Amplified PCR primers include upstream primers as shown in SEQ ID NO:62 and downstream primers as shown in SEQ ID NO:63; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:17 include Upstream primers set forth in SEQ ID NO:64 and downstream primers set forth in SEQ ID NO:65; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:18 include those set forth in SEQ ID NO:66 The upstream primer shown and the downstream primer shown in SEQ ID NO:67; PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:19 include the upstream primer shown in SEQ ID NO:68 and Downstream primers set forth in SEQ ID NO:69; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:20 include the upstream primer set forth in SEQ ID NO:70 and the upstream primer set forth in SEQ ID NO:71 The downstream primers shown; PCR primers for amplification of the nucleotide sequence shown in SEQ ID NO:21 include the upstream primers shown in SEQ ID NO:72 and the downstream primers shown in SEQ ID NO:73; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:22 include the upstream primer set forth in SEQ ID NO:74 and the downstream primer set forth in SEQ ID NO:75; The PCR primers for amplification of the nucleotide sequence shown in NO: 23 include: the upstream primer shown in SEQ ID NO: 76 and the downstream primer shown in SEQ ID NO: 77; The PCR primers for the amplification of the nucleotide sequence include: the upstream primer shown in SEQ ID NO:78 and the downstream primer shown in SEQ ID NO:79; for the nucleotide sequence shown in SEQ ID NO:25 Amplified PCR primers include upstream primers as shown in SEQ ID NO:80 and downstream primers as shown in SEQ ID NO:81; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:26 include Upstream primer set forth in SEQ ID NO:82 and downstream primer set forth in SEQ ID NO:83; amplification against the nucleotide sequence set forth in SEQ ID NO:27 The PCR primers include upstream primers as shown in SEQ ID NO: 84 and downstream primers as shown in SEQ ID NO: 85; PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO: 28 include as SEQ ID NO: 28 The upstream primer set forth in ID NO: 86 and the downstream primer set forth in SEQ ID NO: 87; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO: 29 include set forth in SEQ ID NO: 88 The upstream primers and the downstream primers as shown in SEQ ID NO:89; the PCR primers for amplification of the nucleotide sequence as shown in SEQ ID NO:30 include the upstream primers as shown in SEQ ID NO:90 and as shown in SEQ ID NO:30 Downstream primer set forth in ID NO:91; PCR primers for amplification of the nucleotide sequence set forth in SEQ ID NO:31 include the upstream primer set forth in SEQ ID NO:92 and the upstream primer set forth in SEQ ID NO:93 downstream primers.
  7. 如权利要求4所述的方法，其特征在于，所述步骤1所述待测样品中的食源性致病菌菌体浓度控制在1×10 4～1×10 6cfu/mL范围内。 The method of claim 4, wherein the concentration of food-borne pathogenic bacteria in the sample to be tested in the step 1 is controlled within the range of 1×10 4 to 1×10 6 cfu/mL.
  8. 如权利要求4所述的方法，其特征在于，所述步骤1具体操作如下：在待测样品中加入PMA溶液，将其置于37℃恒温培养箱中避光培养5min，用500W钨丝灯照射10min。该步骤的目的是为了使死菌或细胞膜破损的菌的DNA与PMA交联，使其不能进行PCR扩增。The method according to claim 4, wherein the specific operation of step 1 is as follows: add PMA solution to the sample to be tested, place it in a 37°C constant temperature incubator for 5 min in the dark, and use a 500W tungsten filament lamp Irradiate for 10min. The purpose of this step is to cross-link the DNA of dead bacteria or bacteria with damaged cell membranes to PMA so that PCR amplification cannot be performed.
  9. 如权利要求4所述的方法，其特征在于，所述PMA终浓度为2～16mg/mL。更优选地，所述PMA终浓度为16mg/mL。The method of claim 4, wherein the final concentration of PMA is 2-16 mg/mL. More preferably, the final concentration of PMA is 16 mg/mL.
  10. 如权利要求4所述的方法，其特征在于，所述步骤2提取菌体DNA采用磁珠法提取菌悬液中的DNA。The method according to claim 4, characterized in that, in said step 2, the extraction of bacterial cell DNA adopts a magnetic bead method to extract the DNA in the bacterial suspension.
PCT/CN2021/087080 2020-12-30 2021-04-13 Detection method for live bacteria of standard strain of food-borne pathogenic bacteria having specific molecular target, and use WO2022141945A1 (en)

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CN113388539B (en) * 2020-12-30 2022-05-20 广东省微生物研究所（广东省微生物分析检测中心） Salmonella standard strain containing specific molecular target and detection and application thereof
CN112538544B (en) * 2020-12-30 2022-06-14 广东省微生物研究所（广东省微生物分析检测中心） Detection method and application of food-borne pathogenic bacteria standard strain viable bacteria with specific molecular targets
CN112877448B (en) * 2020-12-30 2022-05-20 广东省微生物研究所（广东省微生物分析检测中心） Bacillus cereus standard strain containing specific molecular target and detection and application thereof
CN112725474B (en) * 2020-12-30 2022-05-20 广东省微生物研究所（广东省微生物分析检测中心） Listeria monocytogenes standard strain containing specific molecular target and detection and application thereof
CN112592993B (en) * 2020-12-30 2022-05-20 广东省微生物研究所（广东省微生物分析检测中心） Staphylococcus aureus standard strain containing specific molecular target and detection and application thereof
CN112646906B (en) * 2020-12-30 2022-06-14 广东省微生物研究所（广东省微生物分析检测中心） Diarrhea-causing escherichia coli standard reference strain containing specific molecular target and detection and application thereof
CN112646907B (en) * 2020-12-30 2022-05-20 广东省微生物研究所（广东省微生物分析检测中心） Vibrio parahaemolyticus standard strain containing specific molecular target and detection and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105543367A (en) * 2016-01-12 2016-05-04 上海海洋大学 Method for simultaneously quantifying two viable pathogenic bacteria in penaeus vanmamei
CN106399489A (en) * 2016-08-31 2017-02-15 北京卓诚惠生生物科技股份有限公司 Fluorescent PCR primer and probe group and kit for detecting living bacteria in clinical sample
CN108330176A (en) * 2017-09-30 2018-07-27 扬州大学 A kind of PCR detection kit of Rapid identification white diarrhea/Salmonella gallinarum
CN112538544A (en) * 2020-12-30 2021-03-23 广东省微生物研究所（广东省微生物分析检测中心） Detection method and application of food-borne pathogenic bacteria standard strain viable bacteria with specific molecular targets

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060246463A1 (en) * 2005-04-20 2006-11-02 Vevea Dirk N Methods and oligonucleotides for the detection of Salmonella SP., E coli 0157:H7, and Listeria monocytogenes
BRPI0710889A2 (en) * 2006-04-24 2011-08-16 Sigma Alimentos Sa De Cv method for multiple and simultaneous detection and quantification of pathogens by real time polymerase chain reaction
CN103667251B (en) * 2012-09-03 2016-03-23 中国科学院上海生命科学研究院 A kind of method of high throughput testing food-borne pathogens
CN103484546B (en) * 2013-09-17 2015-07-01 北京卓诚惠生生物科技有限公司 Fourteen-food-borne pathogenic bacterium multiplex PCR detection primer set and kit
CN104651487B (en) * 2014-11-05 2018-06-15 蔡先全 Detect the kit of food-borne pathogens and multiple fluorescence PCR detection method
CN104726594B (en) * 2015-03-27 2017-05-31 嘉兴市疾病预防控制中心 The heavy fluorescence PCR detection reagent kit of food-borne pathogens five
CN107190079B (en) * 2017-06-30 2020-04-17 北京百康芯生物科技有限公司 Quick detection technology and kit for five food-borne pathogenic bacteria micro-fluidic chip
CN109182567A (en) * 2018-09-27 2019-01-11 军事科学院军事医学研究院环境医学与作业医学研究所 A kind of method of real-time fluorescence quantitative PCR that is while detecting 12 kinds of pathogenic bacterias

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105543367A (en) * 2016-01-12 2016-05-04 上海海洋大学 Method for simultaneously quantifying two viable pathogenic bacteria in penaeus vanmamei
CN106399489A (en) * 2016-08-31 2017-02-15 北京卓诚惠生生物科技股份有限公司 Fluorescent PCR primer and probe group and kit for detecting living bacteria in clinical sample
CN108330176A (en) * 2017-09-30 2018-07-27 扬州大学 A kind of PCR detection kit of Rapid identification white diarrhea/Salmonella gallinarum
CN112538544A (en) * 2020-12-30 2021-03-23 广东省微生物研究所（广东省微生物分析检测中心） Detection method and application of food-borne pathogenic bacteria standard strain viable bacteria with specific molecular targets

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE NUCLEOTIDE 2 September 2002 (2002-09-02), ANONYMOUS : "Sequence 2472 from Patent WO0228891", XP055947995, retrieved from NCBI Database accession no. AX415481 (+ AX415541) *

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