Quadruple Taqman qPCR reagent for detecting food-borne pathogenic bacteria in aquatic products

Abstract

The invention discloses a quadruple Taqman qPCR reagent for detecting food-borne pathogenic bacteria in aquatic products, the food-borne pathogenic bacteria are vibrio parahaemolyticus, vibrio vulnificus, shigella and staphylococcus aureus, and the reagent comprises specific primers and Taqman probes corresponding to the four food-borne pathogenic bacteria. The quadruple Taqman qPCR reagent provided by the invention has the characteristics of high efficiency, accuracy, sensitivity, rapidness and the like, provides an accurate, rapid, efficient and economical means for detecting the four food-borne pathogenic bacteria in the quality safety detection work of aquatic products and processed products thereof, and has important significance for guaranteeing the edible safety of the aquatic products in China.

Description

Technical Field

[0001] The invention belongs to the field of molecular biology detection of foodborne bacteria, and specifically relates to a quadruple Taqman qPCR reagent for detecting foodborne pathogenic bacteria in aquatic products. Background Art

[0002] In recent years, the food safety situation at home and abroad has been poor, and food poisoning incidents caused by pathogenic bacteria contamination have occurred frequently. Foodborne pathogens have become one of the most important factors affecting food safety. With the development of the fishery economy and the advancement of food processing technology, the supply of aquatic products in my country is becoming increasingly abundant, and consumption is also growing. However, aquatic products are extremely susceptible to contamination by pathogenic microorganisms during breeding, fishing, storage, processing, transportation, and sales, and residents in various parts of my country generally have the habit of eating fresh aquatic products such as sashimi, oysters, and raw shrimps. Therefore, detecting foodborne pathogens in aquatic products and their processed products is an important measure to ensure their food safety, which is directly related to the health and life safety of consumers. According to the current national food safety supervision system, Staphylococcus aureus (Sau), Vibrio parahaemolyticus (Vp), Vibrio vulnificus (Vv) and Shigella Castellani (Sc) are all foodborne pathogens of key concern in aquatic products; poisoning incidents caused by these four pathogens on food contamination often manifest as acute gastroenteritis, accompanied by symptoms such as nausea, headache, and low fever, and can cause death in severe cases. Therefore, it is of great significance to establish a rapid detection method for the four foodborne pathogens of Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus in aquatic products and their processed products to ensure the safety of aquatic products in my country.

[0003] Traditional identification methods for foodborne pathogens based on selective culture media and biochemical analysis have defects such as long detection cycle, cumbersome operation, and single detection, which can hardly meet the needs of modern food safety rapid detection. Molecular biological detection technology, such as polymerase chain reaction (PCR), has been widely used in the detection of foodborne pathogens due to its advantages such as high efficiency, sensitivity, and accuracy. Among them, compared with conventional PCR, real-time quantitative PCR (qPCR), especially multiplex qPCR, has gained more widespread attention and application due to its more simplified process, real-time sample quantitative analysis, sample transfer and amplification band visualization without more steps, better specificity and sensitivity, and simultaneous detection of multiple targets. To date, many qPCR kits and detection methods targeting these four pathogens separately, or detecting several of them together with other common foodborne pathogens (such as Escherichia coli, Listeria monocytogenes, etc.) have been reported at home and abroad. For example, the multiplex qPCR kit invented by Cai Xianquan targets the detection of three pathogens: Vibrio parahaemolyticus, Staphylococcus aureus and Listeria monocytogenes; the quintuple qPCR kit invented by He Peiyan targets the detection of Staphylococcus aureus, Listeria monocytogenes, Salmonella, Vibrio parahaemolyticus and Shigella; the seven-fold qPCR kit invented by Shanghai Cebo Biological targets Salmonella, Staphylococcus aureus, Shigella, Escherichia coli, Vibrio parahaemolyticus, Listeria monocytogenes and Bacillus cereus, but there is no research and product dedicated to the simultaneous detection of four important foodborne pathogens: Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus in aquatic products. Therefore, from an application perspective, it is of great significance to develop a rapid, accurate, easy-to-operate, and highly specific quadruple qPCR diagnostic method to achieve the simultaneous identification and detection of Vibrio parahaemolyticus, Vibrio vulnificus, Shigella, and Staphylococcus aureus.

[0004] The quadruple qPCR detection method can simultaneously detect four pathogens, namely, Vibrio parahaemolyticus, Vibrio vulnificus, Shigella (genus) and Staphylococcus aureus, in aquatic products. However, considering that quadruple qPCR requires the addition of four pairs of primers and four probes in one system, in order to ensure that the primers and probes do not affect each other, the primers and probes need to be highly specific to avoid binding or nonspecific amplification between primers and probes. In addition, considering that the situation of nucleic acids in the template is more complicated, and the annealing temperatures required by different primers may be different. Therefore, the establishment of a quadruple Taqman probe qPCR detection method has high research and practical application value, but it is quite difficult to implement. Summary of the invention

[0005] The object of the present invention is to overcome at least one disadvantage of the prior art and to provide a quadruple Taqman qPCR reagent for detecting foodborne pathogens in aquatic products.

[0006] The technical solution adopted by the present invention is:

[0007] In a first aspect, the present invention provides a quadruple Taqman qPCR reagent for detecting foodborne pathogens in aquatic products, wherein the foodborne pathogens are Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus; the reagent comprises specific primers and Taqman probes corresponding to the four foodborne pathogens;

[0008] The primer sequences and Taqman probe sequences for detecting Vibrio parahaemolyticus are: upstream primer Vp-F, downstream primer Vp-R, Taqman probe Vp-P;

[0009] The Vp-F has the sequence shown in SEQ ID NO.1;

[0010] The Vp-R has a sequence as shown in SEQ ID NO.2;

[0011] The Vp-P has a sequence as shown in SEQ ID NO.3;

[0012] The primer sequences and Taqman probe sequences for detecting Vibrio vulnificus are: upstream primer Vv-F, downstream primer Vv-R, Taqman probe Vv-P;

[0013] The Vv-F has the sequence shown in SEQ ID NO.4;

[0014] The Vv-R has the sequence shown in SEQ ID NO.5;

[0015] The Vv-P has the sequence shown in SEQ ID NO.6;

[0016] The primer sequences and Taqman probe sequences for detecting Shigella are: upstream primer Sc-F, downstream primer Sc-R, Taqman probe Sc-P;

[0017] The Sc-F has a sequence as shown in SEQ ID NO.7;

[0018] The Sc-R has a sequence as shown in SEQ ID NO.8;

[0019] The Sc-P has a sequence as shown in SEQ ID NO.9;

[0020] The primer sequences and Taqman probe sequences for detecting Staphylococcus aureus are: upstream primer Sau-F, downstream primer Sau-R, Taqman probe Sau-P, upstream primer Sau-F1, downstream primer Sau-R1, Taqman probe Sau-P1;

[0021] The Sau-F has a sequence as shown in SEQ ID NO.10;

[0022] The Sau-R has a sequence as shown in SEQ ID NO.11;

[0023] The Sau-P has a sequence as shown in SEQ ID NO.12;

[0024] The Sau-F1 has a sequence as shown in SEQ ID NO.13;

[0025] The Sau-R1 has a sequence as shown in SEQ ID NO.14;

[0026] The Sau-P1 has the sequence shown in SEQ ID NO.15.

[0027] In some examples, probes from different strains are labeled with different fluorescent-quencher groups.

[0028] In some examples, the 5' end of the probe Vp-P is labeled with a fluorescent reporter dye FAM, and the 3' end is labeled with a fluorescent quencher group BHQ1;

[0029] The 5' end of the probe Vv-P is labeled with a fluorescent reporter dye Cy5, and the 3' end is labeled with a fluorescent quencher group BHQ2;

[0030] The 5' end of the probe Sc-P is labeled with a fluorescent reporter dye VIC, and the 3' end is labeled with a fluorescent quencher group BHQ1;

[0031] The 5' end of the probe Sau-P is labeled with a fluorescent reporter dye ROX, and the 3' end is labeled with a fluorescent quencher group BHQ2.

[0032] In a second aspect, the present invention provides a quadruple Taqman qPCR kit for detecting foodborne pathogens in aquatic products, wherein the quadruple Taqman qPCR kit comprises the quadruple Taqman qPCR detection reagent described in the first aspect.

[0033] In some examples, the foodborne pathogens are Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus.

[0034] In some examples, the kit further comprises a negative control, a positive control and a lysis solution.

[0035] In some examples, the positive control is a mixture of genomic DNA corresponding to Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus.

[0036] In some examples, the negative control is DEPC water.

[0037] In some examples, the lysate consists of 4.9-5.1 mmol / L tris(hydroxymethylaminomethane) and 0.019-0.121% sodium dodecyl sulfate solution.

[0038] In a third aspect, the present invention provides a detection method of a quadruple Taqman qPCR detection reagent for detecting Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus in aquatic products, comprising the following steps:

[0039] 1) Prepare genomic DNA of the sample to be tested for future use;

[0040] 2) adding the genomic DNA of the sample to be tested as a template to a quadruple qPCR reaction system for amplification, wherein the reaction system includes the quadruple Taqman qPCR kit described in the second aspect;

[0041] 3) Set up the qPCR program and collect the fluorescence signal during the amplification process, and determine the results.

[0042] The beneficial effects of the present invention are:

[0043] 1. Since there is currently a lack of effective methods for simultaneously detecting foodborne pathogens in aquatic products, the present invention, based on the characteristics of important foodborne pathogens in aquatic products, combines multiplex qPCR technology to design primers and probe sets for detecting four foodborne pathogens in aquatic products by multiplex qPCR, including upstream and downstream primers and probes for Vibrio parahaemolyticus, upstream and downstream primers and probes for Vibrio vulnificus, upstream and downstream primers and probes for Shigella, and upstream and downstream primers and probes for Staphylococcus aureus, which correspond to SEQ ID NOs 1, 2 and 3, SEQ ID NOs 4, 5 and 6, SEQ ID NOs 7, 8 and 9, and SEQ ID NOs 10, 11 and 12 in the sequence table, respectively. This multiplex detection method improves detection efficiency and reduces experimental costs.

[0044] 2. The primer-probe combination provided by the present invention targeting Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus has extremely high specificity. The fluorescent signal can be observed in the corresponding fluorescence detection channel only when the target bacteria are contained in the sample to be tested, which effectively avoids the occurrence of false positive or false negative events and makes the detection results more accurate.

[0045] 3. The quadruple Taqman probe qPCR detection method established in the present invention has a high sensitivity, and the detection sensitivity for Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus can reach 2.0×10 2 CFU / mL, and the results have excellent reproducibility. In addition, compared with the traditional biochemical identification based on pure culture, this detection method can complete the entire process of sample detection (including the pre-enrichment process of the sample) within 8 to 11 hours, of which the qPCR reaction time is 1 hour and the manual operation time is 1 hour.

[0046] 4. In summary, the present invention provides a quadruple Taqman qPCR kit and a detection method thereof, which has the characteristics of high efficiency, accuracy, sensitivity, and rapidity, and provides an accurate, rapid, efficient and economical means for detecting the above four foodborne pathogens in the quality and safety inspection of aquatic products and their processed products, which is of great significance for ensuring the food safety of aquatic products in my country. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 This is the amplification curve of the single-plex qPCR method for detecting Vibrio parahaemolyticus.

[0048] Figure 2 This is the amplification curve of Vibrio vulnificus detected by single-plex qPCR method.

[0049] Figure 3 This is the amplification curve of Shigella detected by single-plex qPCR method.

[0050] Figure 4 This is the amplification curve of the single-plex qPCR method for Staphylococcus aureus.

[0051] Figure 5 The results of the sensitivity test of the quadruple Taqman qPCR reaction system for detecting a single pathogen, Vibrio parahaemolyticus.

[0052] Figure 6 The results of the sensitivity test of the quadruple Taqman qPCR reaction system for detecting a single pathogen, Vibrio vulnificus.

[0053] Figure 7 The sensitivity test results of the quadruple Taqman qPCR reaction system for detecting a single pathogenic Shigella.

[0054] Figure 8 The sensitivity test results of the quadruple Taqman qPCR reaction system for detecting a single pathogenic Staphylococcus aureus.

[0055] Fig. 9This is the standard curve corresponding to the sensitivity test of the quadruple Taqman qPCR reaction system for detecting a single pathogen.

[0056] Fig.10 An example of multiplex qPCR detection of four Cronobacter positive samples mixed together.

[0057] Fig.11 This is the amplification curve of Vibrio parahaemolyticus in the sensitivity test of the quadruple Taqman qPCR reaction system for simultaneous detection of four pathogens.

[0058] Fig.12 This is the amplification curve of Vibrio vulnificus in the sensitivity test of the quadruple Taqman qPCR reaction system for simultaneous detection of four pathogens.

[0059] Fig.13 This is the amplification curve of Shigella in the sensitivity test of the quadruple Taqman qPCR reaction system for simultaneous detection of four pathogens.

[0060] Fig.14 This is the amplification curve of Staphylococcus aureus in the sensitivity test of the quadruple Taqman qPCR reaction system for simultaneous detection of four pathogens.

[0061] Fig.15 This is the standard curve corresponding to the sensitivity test of the quadruple Taqman qPCR reaction system for simultaneous detection of four pathogens.

[0062] Fig.16 Figure 1 is a characterization diagram of the anti-interference ability of the quadruple Taqman qPCR reaction system, where Figure (A) is the actual amplification curve of the four target bacteria after the addition of high-density interfering bacteria, and Figure (B) is the Ct value line display corresponding to Figure (A). DETAILED DESCRIPTION

[0063] The following disclosure provides many different embodiments or examples for implementing different solutions of the present invention.

[0064] Example 1

[0065] A quadruple Taqman qPCR detection reagent includes specific qPCR detection primers and corresponding Taqman probes for Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus, as shown in Table 1. Here, in view of the innovativeness of the Staphylococcus aureus target, 2 pairs of primers and probes were specifically designed.

[0066] Table 1 Sequence list of specific qPCR primers and corresponding Taqman probes for four foodborne pathogens

[0067]

[0068]

[0069] Example 2

[0070] The four-plex Taqman qPCR detection method based on the detection reagents described in Example 1 has the following specific steps:

[0071] (1) Crude extraction of DNA from samples to be tested:

[0072] S1) After selecting suspicious colonies and culturing them continuously for 14 hours, 1 mL of the enrichment solution was transferred to a 1.5 mL sterile centrifuge tube, and centrifuged at 6000 r / min for 5 minutes to completely remove the supernatant;

[0073] S2) add 30 μL of lysis buffer prepared by 5 mmol / L sodium dodecyl sulfate (SDS) and 0.02% (m / v) tris (hydroxymethylaminomethane) (Tris-base), use the lysis buffer to fully dissolve and suspend the precipitate at the bottom of the tube, tap the tube wall to eliminate bubbles, and heat at 99°C for 10 min;

[0074] S3) Centrifuge at 12000r / min for 15min. The supernatant is the DNA sample to be tested. Keep it on ice and centrifuge it again if it is not used for a long time. All DNA samples obtained are stored at -20℃ for future use.

[0075] (2) using 4 sets of qPCR primers and their corresponding probes for detection, using the DNA obtained in (1) as a template, using the primers and probes described in Example 1 to perform qPCR amplification, and collecting the fluorescence signal;

[0076] The qPCR reaction system consisted of 12.5 μL 2×qPCR Mix, 0.3, 03, 0.4, 0.4, 0.3, 0.3, 0.4, 0.4 μL of Vp-F, Vp-R, Vv-F, Vv-R, Sc-F, Sc-R, Sau-F / Sau-F1, and Sau-R / Sau-R1, respectively, with an initial concentration of 10 μmol / L, 0.3, 0.4, 0.3, 0.4 μL of Vp-P, Vv-P, Sc-P, and Sau-P / Sau-P1, respectively, with an initial concentration of 10 μmol / L, 2.5 μL of template, and then the total volume was made up to 25 μL with DEPC water;

[0077] The reaction program of qPCR amplification is as follows: the fluorescence channel is set to temperature control, and the program is set to 94°C for 60s; 40 cycles include 95°C for 20s and 59°C for 30s; the fluorescence channel of the qPCR instrument is set to: Channel 1: FAM; Channel 2: Cy5; Channel 3: VIC; Channel 4: ROX; the qPCR instrument automatically collects the fluorescence signal in the set fluorescence channel after each cycle.

[0078] Result determination: Based on the fluorescence signal and Ct value collected in (2), determine whether the sample contains one or more of the four foodborne pathogens. When the Ct value is greater than 35 or the system determines it to be negative, the result is determined to be negative.

[0079] Example 3

[0080] The construction and verification of the reagent described in Example 1 and the detection method described in Example 2, the specific experimental process is as follows:

[0081] 1. Construction and optimization of multiplex qPCR system

[0082] First, the extracted genomic DNA of the target strain was used as a positive control template, DEPC water was used as a blank control, and the four target pathogens were used as negative controls. The corresponding primer probes were used for single-plex qPCR reactions to preliminarily prove the specificity of the designed primer-probe set. Figures 1 to 4 As shown, the primer-probe sets designed for Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus can highly specifically distinguish non-target bacteria. The amplification signal can only be observed in the corresponding fluorescence channel when the corresponding target bacterial DNA is present in the reaction system.

[0083] Subsequently, primers and probes with different final concentrations (set to 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5 μmol / L concentration gradient) were selected for single factor optimization experiments, and the cycle threshold (Ct) and fluorescence signal value (ΔRn) of the amplification curve were comprehensively considered to determine the final single TaqMan qPCR reaction system for the four pathogens. Then, using the extracted genomic DNA of the target strain as the positive control template, on the basis of the single qPCR method, primers of appropriate concentrations were selected for orthogonal experiments to optimize the multiple qPCR reaction system. According to the Ct value of the amplification curve, the change in fluorescence intensity and sensitivity, the optimal conditions for the multiple reaction were selected, so that the four target foodborne pathogens could be detected simultaneously in a single tube system.

[0084] The optimization results of the multiplex qPCR reaction system showed that the concentrations of upstream and downstream primers and probes for Vibrio parahaemolyticus were 0.3 / 03 / 0.3 μmol / L, those for Vibrio vulnificus were 0.4 / 0.4 / 0.4 μmol / L, those for Shigella were 0.3 / 0.3 / 0.3 μmol / L, and those for Staphylococcus aureus (SEQ ID 9-SEQ ID 12) were 0.4 / 0.4 / 0.4 μmol / L, designated as system 1. For the second pair of upstream and downstream primers and probes for Staphylococcus aureus (SEQ ID 13-SEQ ID 15), during the optimization of the multiplex qPCR reaction system (system 2), we found that the primers and probes could not achieve sensitive detection of Staphylococcus aureus: with the same amount of template input, the Ct value would increase by 2 to 3 (Table 2), which means that the sensitivity of subsequent detection would lose an order of magnitude. Therefore, this primer and probe pair was removed from the experiment and was not considered in the future.

[0085] Table 2 Optimization and Ct mean comparison of two multiplex qPCR reaction systems

[0086]

[0087] Note: The bacterial inoculation density of the four target bacteria is 2×10 7 CFU / mL

[0088] 2. Specificity verification of multiplex qPCR system

[0089] In order to verify the specificity of the multiplex qPCR kit detection system constructed by the present invention, the genomic DNA of the target strain and other common non-target bacteria of other genera was extracted as a template based on the method described in Example 2, and a 25 μL detection system was established according to the results of the optimization of Example 2 for multiplex qPCR kit detection, and each template was subjected to 3 repeated reactions. A total of 105 strains of bacteria were verified, including 53 target strains and 52 common non-target bacteria of other species. The target strains are specifically: Vibrio parahaemolyticus (8 standard strains, 10 isolates), Vibrio vulnificus (5 standard strains, 1 isolate), Shigella (11 standard strains, 3 isolates) and Staphylococcus aureus (5 standard strains, 10 isolates). Vibrio parahaemolyticus (ATCC 17802), Vibrio vulnificus (ATCC 27562), Shigella (CMCC (B) 51105) and Staphylococcus aureus (ATCC 25923) were used as positive controls, and DEPC water was used as a negative control.

[0090] The results of the multiplex qPCR kit specificity verification are shown in Table 3. Among a total of 105 bacterial strains (including target bacteria and other common species), only Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus had positive amplification results in their corresponding fluorescence channels. This result shows the high specificity of the multiplex qPCR kit detection system constructed by the present invention.

[0091] Table 3. Strains used for verification of the specificity of the multiplex qPCR kit detection system

[0092]

[0093]

[0094]

[0095] Note: “+” indicates positive result, “–” indicates negative result

[0096] Example 4: Quadruple Taqman qPCR sensitivity experiment

[0097] Vibrio parahaemolyticus (ATCC 17802), Vibrio vulnificus (ATCC 27562), Shigella (CMCC(B)51105) and Staphylococcus aureus (ATCC 25923) were cultured overnight, and the bacterial density of these four pathogens was quantified by plate counting method. The quantitative results showed that the original bacterial density of Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus was 6.0×10 8 CFU / mL, 7.2×10 8 CFU / mL, 4.1×10 8 CFU / mL, 7.8×10 8 CFU / mL.

[0098] 1. Single infection experiment: Take 1 mL of each original bacterial solution and adjust the bacterial density of each bacterium to 2.0×10 8 CFU / mL, 7 gradients of 10-fold dilutions were performed downward (dilution factors: 0, -1, -2, -3, -4, -5, -6, -7). Subsequently, 1 mL of each gradient dilution of each bacterium was taken to extract its genomic DNA based on the method described in Example 2, and multiple qPCR amplification and detection were performed based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2 to explore the sensitivity of the detection method, and each reaction was repeated 3 times.

[0099] 2. Co-infection experiment: Take a certain volume of each target bacteria original bacterial solution and sterile saline and mix them so that the bacterial solution density of each target bacteria in the mixed solution is normalized to 2.0×108 CFU / mL. Then, the normalized mixture was diluted downward by 7 gradients of 10 times. Subsequently, 1 mL of each gradient dilution was taken to obtain genomic DNA based on the method described in Example 2, and multiple qPCR amplification and detection were performed based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2 to explore the sensitivity of the detection method, and each reaction was repeated 3 times.

[0100] The results showed that the kit had a seeding density of 2.0×10 7 Up to 2.0×10 2 CFU / mL (6 gradients) of genomic DNA of Vibrio parahaemolyticus, Vibrio vulnificus, and Staphylococcus aureus, and the inoculation density of 2.0×10 7 Up to 2.0×10 1 When the genomic DNA of Shigella CFU / mL was tested, a good linear relationship was shown (R 2 >0.99); the minimum detection density of Vibrio parahaemolyticus, Vibrio vulnificus and Staphylococcus aureus can be as low as 2.0×10 2 CFU / mL, the minimum detection density of Shigella can be as low as 2.0×10 1 CFU / mL. The standard curves of the multiplex qPCR kit for single pathogen detection and multi-pathogen detection were constructed according to the corresponding Ct mean values. In the single pathogen detection, the slopes of the standard curves of Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus were -3.414, -3.387, -3.099, -3.304, and the corresponding amplification efficiencies were 96%, 97%, 110%, and 101%, respectively; in the multi-pathogen detection, the slopes of the standard curves of Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus were -3.313, -3.439, -3.435, -3.242, and the corresponding amplification efficiencies were 100%, 95%, 95%, and 103%, respectively. The amplification efficiency met the threshold range set by the multiplex qPCR (80%-120%), indicating that the quadruple Taqman qPCR method has good amplification performance. At the same time, it also shows that the kit has the ability to detect single-infection samples and multi-infection samples. The results are detailed in Table 4 and Figures 5 to 9 (single pathogen infection) and Table 5 and Figures 10 to 13 (Multi-pathogen infection).

[0101] Table 4 Sensitivity test results of the quadruple Taqman qPCR system for detecting a single pathogen

[0102]

[0103] Note: “–” indicates a negative result, the same below.

[0104] Table 5 Sensitivity test results of the quadruple Taqman qPCR detection method for simultaneous detection of four target pathogens

[0105]

[0106] Example 5: Stability Test

[0107] Using the normalized concentration gradient premix in Example 4 as the input DNA template, based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2, a repeatability experiment (repeated 8 times) was carried out on the "co-infection sensitivity" in Example 4, and the stability of the method was detected according to the coefficient of variation (CV). Coefficient of variation = standard deviation / mean.

[0108] The results are shown in Table 6. When the quadruple qPCR was used to detect the normalized concentration gradient premix, the CV (%) values ​​between 8 replicates of each detection gradient were basically less than 2%, and very few were between 2% and 3%, meeting the set threshold requirement for precision of the in vitro diagnostic kit (<15%), indicating that the system has good stability.

[0109] Table 6 Stability evaluation of quadruple Taqman qPCR for simultaneous detection of four target pathogens

[0110]

[0111]

[0112]

[0113]

[0114] Example 6: Anti-interference experiment

[0115] According to the characteristics of aquatic products, five foodborne pathogens, including Salmonella typhimurium (ATCC 14028), Listeria monocytogenes (ATCC19115), Escherichia coli O157:H7 (NCTC 12900), Aeromonas hydrophila (CMCC(B)10506), and Vibrio alginolyticus (ATCC33787), were selected as interfering bacteria. The five selected interfering bacteria and four target bacteria, including Vibrio parahaemolyticus (ATCC 17802), Vibrio vulnificus (ATCC 27562), Shigella (CMCC(B)51105), and Staphylococcus aureus (ATCC 25923), were cultured overnight, and the bacterial density of these nine pathogens was quantified by plate counting method. The quantitative results showed that the original bacterial liquid densities of Salmonella, Listeria monocytogenes, Escherichia coli O157:H7, Aeromonas hydrophila and Vibrio alginolyticus were 3.64×10 8 , 2.58×10 9 , 1.30×10 8 , 3.21×10 8 4.08×10 8 CFU / mL, the original bacterial liquid densities of Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus were 2.13×10 8 , 3.50×10 8 , 8.75×10 8 ,8.82×10 8 CFU / mL. Then, the target bacteria solution was diluted downward in three gradients of 10 times (dilution factors: 0, -1, -2, -3), and 2 mL was taken from the four target bacteria dilutions (dilution factor of -3) and mixed. After that, 1 mL was taken from each of the five interfering bacteria stock dilutions (dilution factor of -1) of Escherichia coli O157:H7, Salmonella typhimurium, Vibrio alginolyticus, Aeromonas hydrophila, and Listeria monocytogenes and added to 1 mL of the prepared target bacteria dilution mixture for equal mixing. After sufficient shaking and mixing, 1 mL was taken to extract DNA based on the method described in Example 2, and multiple qPCR amplification and detection were performed based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2. The results are shown in Table 7 and Fig.14 As shown, high-density interfering bacteria (10 8 The addition of 2% (CFU / mL) did not significantly interfere with the detection of target bacteria, and the Ct values ​​were basically the same.

[0116] Table 7 Ct values ​​of quadruple qPCR after addition of high-density non-target bacteria

[0117]

[0118] Note: The positive control is the template DNA obtained by diluting the target bacteria dilution solution with a dilution factor of -3 down by 2 times.

[0119] Example 7: Artificial contamination sample test

[0120] (1) Preparation of gradient dilutions of bacterial solutions: Vibrio parahaemolyticus (ATCC 17802), Vibrio vulnificus (ATCC 27562), Shigella (ATCC 50115) and Staphylococcus aureus (ATCC 25923) were cultured overnight, and the bacterial density of these four pathogens was quantified by plate count method, and six gradients of 10-fold dilutions were performed downward (dilution factors: 0, -1, -2, -3, -4, -5, -6).

[0121] (2) Simulation sample preparation: Aseptic operation, take 25g of sterilized shrimp meat, add 225ml of 3.5% sterile NaCl solution, homogenize, take 9ml and add to 90ml of corresponding culture matrix: 3% Nacl alkaline peptone water (Vibrio parahaemolyticus), PNCC (Vibrio vulnificus), GN (Shigella), 7.5% Nacl broth (Staphylococcus aureus). Take 1mL of each gradient of the gradient dilution bacterial suspension prepared in (1) and add it to the corresponding enrichment solution, take the last 4 gradients (dilution factors: -3, -4, -5, -6) and culture and enrich the bacteria in a 37°C shaker (155r / min) (time setting: 3, 6, 9h). After completion, take 1mL of each target bacteria from each gradient to extract DNA as a template, and perform multiplex qPCR amplification and detection based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2.

[0122] When the dilution factor is -6, the concentration of bacteria in the simulated sample is about 1-10 CFU / mL, which is equivalent to 1-10 CFU / g of bacteria in the sample. The amplification results of artificial contaminated samples are shown in Table 8. In the absence of bacterial culture, the positive detection limit of Vibrio parahaemolyticus and Vibrio vulnificus in the simulated sample is 10 2 CFU / mL (dilution factor is -4), the positive detection limit of Shigella in the simulated sample is 10 1 CFU / mL (dilution factor is -5), the positive detection limit of Staphylococcus aureus in the simulated sample is 10 3 CFU / mL dilution factor is -3); after 6 hours of bacterial growth, the positive detection limits of Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus in the simulated samples can reach 10 0CFU / mL (dilution factor is -6). However, for Shigella and Staphylococcus aureus, it is better to extend the bacterial enrichment time to 9 hours. In general, compared with the detection cycle of at least 18 hours based on the traditional method, this method can complete the detection results within 8 to 11 hours, including 6 to 9 hours of bacterial enrichment, 1 hour of qPCR and 1 hour of manual operation time, which has a strong time cost-effectiveness.

[0123] Table 8 Results of quadruple qPCR after enrichment of artificially contaminated samples

[0124]

[0125]

[0126]

[0127]

[0128] Example 8: Multiplex qPCR for clinical testing of aquatic products

[0129] The established multiplex qPCR was applied to detect 14 aquatic products purchased from local supermarkets and farmers' markets, including salmon (n=2), tilapia (n=3), mud shrimp (n=1), sand shrimp (n=1), prawns (n=1), kelp (n=1), cuttlefish balls (n=1), squid (n=1), squid tentacles (n=1), oysters (n=1), and shrimp (n=1). Specific method: 25g of each sample was weighed and added to the corresponding enrichment solution described in Example 7. After homogenization, 1mL was extracted from it after incubation at 37°C for 6-9h as an amplification template, and multiplex qPCR amplification and detection were performed based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2. At the same time, these 14 aquatic products were tested for Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus in accordance with GB 4789.7-2013, GB4789.44-2020, GB4789.5-2012 and GB4789.10-2016, and the test results of the two methods were compared.

[0130] The results showed that, using this multiplex qPCR kit, 10 of the 14 aquatic products were positive for Vibrio parahaemolyticus, 7 for Vibrio vulnificus, 4 for Staphylococcus aureus, and 0 for Shigella. On the other hand, the national standard method detected 10 Vibrio parahaemolyticus, 6 for Vibrio vulnificus, 4 for Staphylococcus aureus, and 0 for Shigella. In general, the positive samples detected by the traditional method were consistent with the qPCR test results, but the positive detection rate of foodborne pathogens detected by the qPCR method was slightly higher than that of the traditional method (Table 9). The reason for the inconsistent test results can be attributed to the fact that the traditional detection method relies on obtaining pure bacterial cultures, and the target bacteria are likely to be missed during the purification culture process.

[0131] Table 9 Comparison of the detection results of 4 target bacteria in 14 aquatic products by quadruple Taqman qPCR and national standard identification method

[0132]

[0133] Note: “+” indicates a positive result, and “-” indicates a negative result.

[0134] In general, the above embodiments show and describe the basic principles and features of the present invention. The results show that the kit of the present invention has the characteristics of good accuracy, repeatability and high sensitivity, is suitable for rapid detection and identification of four important foodborne pathogens in aquatic products, and has strong market application and promotion potential.

[0135] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A quadruple Taqman qPCR reagent for detecting foodborne pathogens in aquatic products, characterized in that: The foodborne pathogens are Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus; the reagents include specific primers and Taqman probes corresponding to the four foodborne pathogens; The primer sequences and Taqman probe sequences for detecting Vibrio parahaemolyticus are: upstream primer Vp-F, downstream primer Vp-R, Taqman probe Vp-P; The Vp-F has the sequence shown in SEQ ID NO.1; The Vp-R has a sequence as shown in SEQ ID NO.2; The Vp-P has a sequence as shown in SEQ ID NO.3; The primer sequences and Taqman probe sequences for detecting Vibrio vulnificus are: upstream primer Vv-F, downstream primer Vv-R, Taqman probe Vv-P; The Vv-F has the sequence shown in SEQ ID NO.4; The Vv-R has the sequence shown in SEQ ID NO.5; The Vv-P has the sequence shown in SEQ ID NO.6; The primer sequences and Taqman probe sequences for detecting Shigella are: upstream primer Sc-F, downstream primer Sc-R, Taqman probe Sc-P; The Sc-F has a sequence as shown in SEQ ID NO.7; The Sc-R has a sequence as shown in SEQ ID NO.8; The Sc-P has a sequence as shown in SEQ ID NO.9; The primer sequences and Taqman probe sequences for detecting Staphylococcus aureus are: upstream primer Sau-F, downstream primer Sau-R, Taqman probe Sau-P, upstream primer Sau-F1, downstream primer Sau-R1, Taqman probe Sau-P1; The Sau-F has a sequence as shown in SEQ ID NO.10; The Sau-R has a sequence as shown in SEQ ID NO.11; The Sau-P has a sequence as shown in SEQ ID NO.12; The Sau-F1 has a sequence as shown in SEQ ID NO.13; The Sau-R1 has a sequence as shown in SEQ ID NO.14; The Sau-P1 has the sequence shown in SEQ ID NO.

15.

2. The quadruple Taqman qPCR reagent according to claim 1, characterized in that Probes for different strains are labeled with different fluorescent-quenching groups.

3. The quadruple Taqman qPCR reagent according to claim 1 or 2, characterized in that: The 5' end of the probe Vp-P is labeled with a fluorescent reporter dye FAM, and the 3' end is labeled with a fluorescent quencher group BHQ1; The 5' end of the probe Vv-P is labeled with a fluorescent reporter dye Cy5, and the 3' end is labeled with a fluorescent quencher group BHQ2; The 5' end of the probe Sc-P is labeled with a fluorescent reporter dye VIC, and the 3' end is labeled with a fluorescent quencher group BHQ1; The 5' end of the probe Sau-P is labeled with a fluorescent reporter dye ROX, and the 3' end is labeled with a fluorescent quencher group BHQ2.

4. A quadruple Taqman qPCR kit for detecting foodborne pathogens in aquatic products, characterized in that: The quadruple Taqman qPCR kit comprises the quadruple Taqman qPCR detection reagent according to any one of claims 1 to 3.

5. The quadruple Taqman qPCR kit according to claim 4, characterized in that The foodborne pathogenic bacteria are Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus.

6. The quadruple Taqman qPCR kit according to claim 4, characterized in that The kit also includes a negative control substance, a positive control substance and a lysate.

7. The quadruple Taqman qPCR kit according to claim 6, characterized in that The positive control substance is a mixture of genomic DNA corresponding to Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus.

8. The quadruple Taqman qPCR kit according to claim 6, characterized in that The negative control substance is DEPC water.

9. The quadruple Taqman qPCR kit according to claim 6, characterized in that: The lysate comprises 4.9-5.1 mmol / L tris(hydroxymethylaminomethane) and 0.019-0.121% sodium dodecyl sulfate solution.

10. A detection method for detecting Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus in aquatic products using a quadruple Taqman qPCR detection reagent, characterized in that: The following steps are involved: 1) Prepare genomic DNA of the sample to be tested for backup; 2) adding the genomic DNA of the sample to be tested as a template to a quadruple qPCR reaction system for amplification, wherein the reaction system comprises the quadruple Taqman qPCR kit according to claim 4; 3) Set up the qPCR program and collect the fluorescence signal during the amplification process, and determine the results.

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