Multiple qPCR (quantitative polymerase chain reaction) kit for detecting food-borne pathogenic bacteria of aquatic products and application of multiple qPCR kit

Abstract

The invention belongs to the technical field of biological detection, and discloses a multiple qPCR (quantitative polymerase chain reaction) kit for detecting food-borne pathogenic bacteria of aquatic products and application of the multiple qPCR kit. The kit comprises primers and a probe group for detecting vibrio parahaemolyticus, listeria monocytogenes and salmonella, and the sequences of the primers and the probe group are as shown in SEQ ID NO.1-9. According to the invention, probes with different fluorescence labels are utilized to realize simultaneous detection of three food-borne pathogenic bacteria of vibrio parahaemolyticus, listeria monocytogenes and salmonella in aquatic products. The method has the characteristics of short time consumption, good specificity, high sensitivity, high economic benefit and the like, has important significance on molecular diagnosis of the food-borne pathogenic bacteria in the aquatic products, and meets the detection requirements of simultaneously detecting the three food-borne pathogenic bacteria in the aquatic products.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological detection, and particularly relates to a multiplex qPCR kit for detecting foodborne pathogenic bacteria in aquatic products and its application. Background Art

[0002] Foodborne pathogenic bacteria in aquatic products are one of the important causes of human food poisoning. Especially, ready-to-eat aquatic products such as sashimi are easily infected by environmental and human host bacteria. Since they are not heat-treated before consumption, there is a risk of foodborne pathogenic bacteria infection.

[0003] Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella are common foodborne pathogenic bacteria in aquatic products.

[0004] As a Gram-negative halophilic bacterium, Vibrio parahaemolyticus is widely distributed in aquatic products, and its pathogenicity is related to human acute gastroenteritis. Human consumption of aquatic products containing Vibrio parahaemolyticus can cause food poisoning, and the symptoms include headache, vomiting, and acute diarrhea, etc. At the same time, it is also a zoonotic pathogen.

[0005] Listeria monocytogenes is a Gram-positive bacterium. It can move within a wide temperature range and can quickly adapt to environmental changes to survive and reproduce. Listeria monocytogenes in many food types can grow and survive under conditions of refrigeration temperature, low pH, high salt, and low water activity. Mild infection with Listeria monocytogenes can cause gastroenteritis, accompanied by symptoms such as fever, diarrhea, nausea, and vomiting. Severe human listeriosis has a high hospitalization rate and fatality rate, including symptoms such as sepsis, meningitis, stillbirth, and miscarriage.

[0006] Salmonella is a Gram-negative bacillus of the Enterobacteriaceae family and is widely distributed in nature. Salmonella spreads diseases through contaminated water, vegetables, and seafood. It can cause diseases of varying degrees from gastroenteritis to typhoid fever. The symptoms are mild, such as high fever, diarrhea, vomiting, and headache, and severe symptoms can lead to sepsis and even death.

[0007] Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella are all key objects of concern in the national food safety standard "Limit of Pathogenic Bacteria in Prepackaged Foods" GB 29921-2021.

[0008] Traditional culture method detection requires a series of steps such as enrichment, streaking separation on selective medium, purification by streaking on a plate, and picking morphologically suspected single colonies for biochemical identification. The traditional culture detection method is a reliable system in food diagnosis and is the gold standard for detecting foodborne pathogenic bacteria in China. However, the traditional detection method is laborious and time-consuming. In the detection of multiple pathogenic bacteria, the traditional culture method is very cumbersome, requires the analysis of a large number of samples, and has a large work intensity. Therefore, there is an urgent need for a rapid detection method for multiple foodborne pathogenic bacteria in aquatic products in the same reaction.

[0009] qPCR is a mature molecular detection technology. Multiplex TaqMan qPCR detection can quickly achieve the simultaneous detection of multiple pathogens by measuring the multi-channel fluorescence signals generated by specific multi-label probes. Summary of the Invention

[0010] Aiming at the deficiencies of existing traditional detections, the purpose of the present invention is to establish a multiplex real-time fluorescence PCR detection system to achieve the rapid detection of three kinds of foodborne pathogenic bacteria in aquatic products, save detection time, save manpower and material resources, and simplify the detection process. The present invention evaluates the specificity, standard curve, anti-interference ability, sensitivity of artificially simulated samples and actual samples of the established multiplex qPCR detection system. This detection method has the characteristics of strong specificity, high sensitivity and fast detection speed.

[0011] On the one hand, the present invention provides a primer-probe set for detecting foodborne pathogenic bacteria in aquatic products, and the primer-probe set includes:

[0012] Primers and probes for detecting Vibrio parahaemolyticus, the primer sequences are shown in SEQ ID NO.1-2, and the probe sequence is shown in SEQ ID NO.3;

[0013] Primers and probes for detecting Listeria monocytogenes, the primer sequences are shown in SEQ ID NO.4-5, and the probe sequence is shown in SEQ ID NO.6;

[0014] Primers and probes for detecting Salmonella, the primer sequences are shown in SEQ ID NO.7-8, and the probe sequence is shown in SEQ ID NO.9;

[0015] The 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quenching group.

[0016] Preferably, the 5' end of the Vibrio parahaemolyticus detection probe is modified with the reporter group Texas Red, and the 3' end is modified with the quenching group BHQ2; the 5' end of the Listeria monocytogenes detection probe is modified with the reporter group Cy5, and the 3' end is modified with the quenching group BHQ2; the 5' end of the Salmonella detection probe is modified with the reporter group FAM, and the 3' end is modified with the quenching group BHQ1.

[0017] On the other hand, the present invention also provides the application of the above primer-probe set in the preparation of a multiplex qPCR detection kit for Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella.

[0018] On the other hand, the present invention also provides a multiplex qPCR detection kit for Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella, which contains the above primer-probe set.

[0019] On the other hand, the present invention also provides a detection method for simultaneously detecting Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella in aquatic products based on multiplex qPCR technology, which comprises the following steps:

[0020] (1) Collect samples for enrichment culture;

[0021] (2) Extract genomic DNA from the enrichment broth;

[0022] (3) Perform multiplex qPCR amplification using the above primer-probe set and collect fluorescence signals;

[0023] (4) Determine whether Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella are detected through fluorescence signals.

[0024] Preferably, the enrichment culture in step (1) is specifically: place the collected samples in 3% sodium chloride peptone water, LB1 enrichment broth and alkaline peptone water respectively for enrichment culture for 12 h.

[0025] More preferably, the enrichment culture in step (1) is specifically: place 25 g of the collected samples in 225 mL of 3% sodium chloride peptone water, LB1 enrichment broth and alkaline peptone water respectively for enrichment culture for 12 h.

[0026] Preferably, the multiplex qPCR reaction system in step (3) is: 2×Probe qPCR SuperMix 5 μL; the forward and reverse primer concentrations of Vibrio parahaemolyticus are 0.07 μmol / L, and the probe concentration is 0.3 μmol / L; the forward and reverse primer concentrations of Listeria monocytogenes are 0.2 μmol / L, and the probe concentration is 0.1 μmol / L; the forward and reverse primer concentrations of Salmonella are 0.4 μmol / L, and the probe concentration is 0.1 μmol / L; 1 μL of template DNA for each of the three bacteria, and make up to 10 μL with RNase-free ddH2O.

[0027] Preferably, the multiplex qPCR reaction program in step (3) is: 50°C, 2 min; pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing and extension at 65°C for 60 s, 40 cycles.

[0028] Preferably, in step (4), when collecting fluorescence signals, when the Cq value is less than 35, the detection result is judged as positive, and when the Cq value is greater than 35, the detection result is judged as negative.

[0029] On the other hand, the present invention also provides the application of the above primer-probe set, kit or detection method in detecting Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella in aquatic products.

[0030] The present invention breaks through the limitations of single fluorescence PCR detection, realizes the simultaneous detection of Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella in aquatic products by real-time fluorescence PCR single-tube reaction, and has the characteristics of rapidity, specificity, economy, etc. It reduces the labor intensity for experiments with a large demand for sample volume and reduces the detection cost of each sample. In the artificial simulated sample detection experiment, the detection limit of Vibrio parahaemolyticus reaches 6.30×10 0 CFU / mL, the detection limit of Listeria monocytogenes reaches 6.13×10 2 CFU / mL, and the detection limit of Salmonella reaches 2.44×10 1 CFU / mL. Description of the Drawings

[0031] Figure 1 It is the specific detection result of single qPCR of Vibrio parahaemolyticus, and label 1 is Vibrio parahaemolyticus.

[0032] Figure 2 It is the specific detection result of single qPCR of Listeria monocytogenes, and label 1 is Listeria monocytogenes.

[0033] Figure 3 It is the specific detection result of single qPCR of Salmonella, and label 1 is Salmonella.

[0034] Figure 4 It is the specific detection result of multiplex qPCR, label 1 is Vibrio parahaemolyticus, label 2 is Listeria monocytogenes, and label 3 is Salmonella.

[0035] Figure 5 It is the standard curve of the multiplex qPCR detection system.

[0036] Figure 6 It is the detection result of the multiplex qPCR anti-interference experiment.

[0037] Figure 7 It is the detection result of the multiplex qPCR artificial simulated sample sensitivity experiment. Labels a, b and c respectively represent the amplification curves of the sensitivities of artificial simulated samples of Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella. Detailed Embodiments

[0038] To further illustrate the technical means and effects adopted by the present invention, the following further explains the technical solution of the present invention through specific embodiments, but the specific implementable content of the present invention is not limited within the scope of the embodiments.

[0039] The materials used in the following embodiments are not limited to the following list, and other similar materials can be used for substitution. For instruments without specific conditions specified, they are in accordance with conventional conditions or the conditions recommended by the manufacturer. Those skilled in the art should master the relevant knowledge of using conventional materials and instruments.

[0040] Experimental reagents: Probe qPCR SuperMix containing UDG (HKE106 - 01A); Probe and primer synthesis (BGI); The liquid media in the following examples: 3% sodium chloride LB broth (for culturing Vibrio parahaemolyticus), LB broth (for culturing Listeria monocytogenes and Salmonella), product number: 028320.

[0041] Experimental instruments: Fluorescent quantitative PCR instrument ( 96).

[0042] Example 1 Design and verification of primers and probes

[0043] 1) Primer and probe design

[0044] Based on the specific nucleic acid sequences of the three bacteria, specific primers and probes were designed using Primer Express software in the highly conserved sequences of Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella respectively. The nucleotide sequences of the specific primers and probes for the three foodborne pathogenic bacteria in aquatic products are shown in SEQ ID NO.1 - 9, as shown in Table 1 below:

[0045] Table 1 Different primer and probe sequences

[0046]

[0047] 2) Specificity verification of primers and probes

[0048] Specificity verification of the qPCR detection system

[0049] Take out Vibrio parahaemolyticus, Listeria monocytogenes, Salmonella, Escherichia coli, Cronobacter, Yersinia enterocolitica, Staphylococcus aureus, Campylobacter jejuni, Staphylococcus epidermidis, and Bacillus cereus from the - 80°C refrigerator for thawing and activation. Culture the strains to the logarithmic growth phase and extract nucleic acids using a DNA extraction kit. According to the following Table 2 - 4, each detection system is equipped with a single - detection system for qPCR detection.

[0050] Table 2 qPCR detection system for Vibrio parahaemolyticus

[0051]

[0052] Table 3 qPCR detection system for Listeria monocytogenes

[0053]

[0054] Table 4 qPCR detection system for Salmonella

[0055]

[0056] The single qPCR amplification reaction procedure is as follows: 50°C for 2 min; pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing and extension at 65°C for 1 min, for 40 cycles. The detection results are as Figures 1-3 shown. Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella can all be detected in their respective single qPCR systems, and no other non-target bacteria are detected. The experimental results show that the primers and probes have strong specificity.

[0057] Based on the optimization of the single qPCR, the multiplex qPCR detection system was optimized. According to the fluorescence signal intensity and Cq value, the system in Table 5 was selected as the final multiplex qPCR reaction system. Configure the multiplex detection system for qPCR amplification. The qPCR amplification reaction procedure is as follows: 50°C for 2 min; pre-denaturation at 94°C for 2 min; denaturation at 94°C for 15 s, annealing and extension at 65°C for 1 min, for 40 cycles. The detection results are as Figure 4 shown. The three pathogenic bacteria in aquatic products can all be detected in the multiplex qPCR detection system, and the detection system has strong specificity.

[0058] Table 5 Multiplex qPCR Detection System

[0059]

[0060] Example 2 Standard Curve of Multiplex qPCR Detection System

[0061] Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella were cultured overnight at 37°C in liquid medium respectively, diluted 10-fold gradient with sterilized normal saline, DNA of different bacterial liquid concentrations was extracted, and the Cq values of multiplex qPCR detection were collected to draw the standard curve. The detection results are as Figure 5 shown. There is a good linear relationship between the logarithm of the three bacterial liquid concentrations and the Cq values, and their correlation coefficients R 2 are 0.9855, 0.9920, and 0.9867 respectively.

[0062] Example 3 Multiplex qPCR Anti-interference Experiment

[0063] Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella were cultured overnight in liquid medium respectively, nucleic acids were extracted using a DNA extraction kit, and the templates of single bacteria and the templates of three bacteria were added to different reaction tubes of the multiplex qPCR detection system for qPCR amplification, and the experiment was repeated 3 times. Differential analysis was performed according to the Cq values. The anti-interference results are as Figure 6 shown. There is no significant difference in the Cq values of single strain detection and mixed bacteria detection in the multiplex detection system (P>0.05), indicating that there is no mutual influence between the Cqs of the three bacteria in this detection system, and this detection system has strong anti-interference ability.

[0064] Sensitivity Experiment of Artificial Simulated Samples for Multiplex qPCR Detection in Example 4

[0065] Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella were cultured overnight at 37°C in a liquid medium, diluted 10-fold using sterilized deionized water, and 1 g of shrimp was inoculated with different concentrations of the bacterial solution. The shrimp were then incubated in 3% sodium chloride peptone water, LB1 enrichment broth, and alkaline peptone water for 12 hours at a 10-fold dilution, and the DNA of the simulated samples was extracted for qPCR detection. The 3% sodium chloride peptone water, LB1 enrichment broth, and alkaline peptone water used in this experiment were all from Huankai Microbial Technology Co., Ltd., with the product numbers C25015G1, CP0500A, and 023130, respectively. The detection results are as Figure 7 shown. The results of the artificial contamination experiment showed that the detection limit of Vibrio parahaemolyticus reached 6.30×10 0 CFU / mL, the detection limit of Listeria monocytogenes reached 6.13×10 2 CFU / mL, and the detection limit of Salmonella reached 2.44×10 1 CFU / mL.

[0066] Example 5 Multiplex qPCR Detection of Actual Samples

[0067] A total of 60 aquatic product samples were purchased from the Guangdong market and supermarkets, and the isolation and identification of 3 target bacteria were carried out with reference to the national food microbiological inspection standards GB4789.7-2013, GB 4789.30-2016, and GB 4789.4-2024. At 12 h of enrichment, 1 mL of the enrichment broth was taken to extract the DNA of the sample as the detection template for multiplex qPCR detection, and the results are shown in Table 6. The multiplex qPCR detection results of the present invention were consistent with the national standard detection results.

[0068] Table 6 Detection Results of Actual Samples

[0069]

[0070]

[0071]

[0072] The results of the above examples show that the multiplex qPCR detection kit for aquatic products of the present invention has the characteristics of high sensitivity, strong specificity, and good anti-interference ability. This detection method meets the conditions for detecting multiple pathogenic bacteria in complex food matrices.

[0073] The above are only the preferred embodiments of the present invention. The above preferred embodiments should not be construed as limiting the present invention. The protection scope of the present invention shall be subject to the scope defined by the claims. Those skilled in the art of this technology can also make several improvements and refinements without departing from the spirit and scope of the present invention, and these improvements and refinements should also be regarded as within the protection scope of the present invention.

Claims

1. A primer-probe set for detecting foodborne pathogenic bacteria in aquatic products, characterized in that, The primer-probe set includes: Primers and a probe for detecting Vibrio parahaemolyticus, with the primer sequences shown in SEQ ID NO.1-2 and the probe sequence shown in SEQ ID NO.3; Primers and a probe for detecting Listeria monocytogenes, with the primer sequences shown in SEQ ID NO.4-5 and the probe sequence shown in SEQ ID NO.6; Primers and a probe for detecting Salmonella, with the primer sequences shown in SEQ ID NO.7-8 and the probe sequence shown in SEQ ID NO.9; The 5'-end of the probe is labeled with a fluorescent reporter group, and the 3'-end is labeled with a fluorescent quenching group.

2. The primer-probe set according to claim 1, wherein The 5'-end of the Vibrio parahaemolyticus detection probe is modified with the reporter group Texas Red, and the 3'-end is modified with the quenching group BHQ2; the 5'-end of the Listeria monocytogenes detection probe is modified with the reporter group Cy5, and the 3'-end is modified with the quenching group BHQ2; the 5'-end of the Salmonella detection probe is modified with the reporter group FAM, and the 3'-end is modified with the quenching group BHQ1.

3. The application of the primer-probe set according to claim 1 or 2 in the preparation of a multiplex qPCR detection kit for Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella.

4. A multiplex qPCR detection kit for Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella, characterized in that, Comprising the primer-probe set according to claim 1 or 2.

5. A detection method for simultaneously detecting Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella in aquatic products based on multiplex qPCR technology, characterized in that, Including the following steps: (1) Collect samples for enrichment culture; (2) Extract the genomic DNA of the enrichment broth; (3) Use the primer-probe set according to claim 1 or 2 for multiplex qPCR amplification and collect fluorescence signals; (4) Determine whether Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella are detected through the fluorescence signals.

6. The detection method according to claim 5, characterized in that, The enrichment culture in step (1) is specifically: Place the collected samples in 3% sodium chloride peptone water, LB1 enrichment broth, and alkaline peptone water for enrichment culture for 12 h.

7. The detection method according to claim 5, wherein The multiplex qPCR reaction system in step (3) is: 2×Probe qPCR SuperMix 5 μL; the forward and reverse primer concentrations of Vibrio parahaemolyticus are 0.07 μmol / L, and the probe concentration is 0.3 μmol / L; the forward and reverse primer concentrations of Listeria monocytogenes are 0.2 μmol / L, and the probe concentration is 0.1 μmol / L; the forward and reverse primer concentrations of Salmonella are 0.4 μmol / L, and the probe concentration is 0.1 μmol / L; 1 μL of the template DNA of each of the three bacteria, and make up to 10 μL with RNase-free ddH2O.

8. The detection method according to claim 5, characterized in that The multiplex qPCR reaction program in step (3) is: 50 °C, 2 min; pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 15 s, annealing and extension at 65 °C for 60 s, 40 cycles.

9. The detection method according to claim 5, wherein When collecting fluorescence signals, when the Cq value is less than 35, the test result is judged as positive, and when the Cq value is greater than 35, the test result is judged as negative.

10. The application of the primer-probe set according to claim 1, the kit according to claim 4, or the method according to claim 5 in the detection of Vibrio parahaemolyticus, Listeria monocytogenes, and Salmonella in aquatic products.

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