A quadruple Taqman qPCR reagent for detecting foodborne pathogenic bacteria in aquatic products

Abstract

This invention discloses a quadruple Taqman qPCR reagent for detecting foodborne pathogens in aquatic products. The foodborne pathogens are Vibrio parahaemolyticus, Vibrio vulnificus, Shigella, and Staphylococcus aureus. The reagent includes specific primers and Taqman probes corresponding to the four foodborne pathogens. This invention provides a quadruple Taqman qPCR reagent with high efficiency, accuracy, sensitivity, and speed, offering an accurate, rapid, efficient, and economical method for detecting the above four foodborne pathogens in the quality and safety testing of aquatic products and their processed products. This is of great significance for ensuring the safety of aquatic products for consumption in my country.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of molecular biology detection of foodborne bacteria, and particularly relates to a quadruple Taqman qPCR reagent for detecting foodborne pathogenic bacteria in aquatic products. BACKGROUND

[0002] In recent years, the food safety situation at home and abroad is not good, and food poisoning events caused by pathogenic bacteria pollution frequently occur. Foodborne pathogenic bacteria have become one of the most important factors affecting food safety. With the development of fishery economy and the progress of food processing technology, the supply of aquatic products in China is increasing, and the consumption is also growing. However, aquatic products are extremely susceptible to pathogenic microorganism contamination at the stages of breeding, fishing, storage, processing, transportation, and sales, and residents in various parts of China generally have the habit of eating fresh aquatic products such as raw fish slices, raw oysters, and raw shrimps. Therefore, detecting foodborne pathogenic bacteria in aquatic products and their processed products is an important measure to ensure their edible 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 pathogenic bacteria that are focused on in aquatic products; these four pathogenic bacteria often cause acute gastroenteritis and symptoms such as nausea, headache, low fever, and even death in food contamination poisoning events. Therefore, establishing a rapid detection method for the four foodborne pathogenic bacteria, Vibrio parahaemolyticus, Vibrio vulnificus, Shigella Castellani, and Staphylococcus aureus in aquatic products and their processed products is of great significance to ensure the edible safety of aquatic products in China.

[0003] Traditional identification methods of foodborne pathogens based on selective culture medium and biochemical analysis have defects such as long detection period, complicated operation and single detection, which are difficult to meet the needs of modern food safety rapid detection. Molecular biology detection techniques, such as polymerase chain reaction (PCR), have been widely used in the detection of foodborne pathogens due to their high efficiency, sensitivity and accuracy. Compared with conventional PCR, real-time quantitative PCR (qPCR), especially multiplex qPCR, has gained more attention and application due to its more simplified process, real-time sample quantitative analysis, no need for more steps of sample transfer and amplification band visualization, more excellent specificity and sensitivity, and simultaneous detection of multiple targets. So far, many qPCR kits and detection methods targeting these four pathogens or several of them and other common foodborne pathogens (such as E. coli and Listeria monocytogenes) have been reported, for example, Cai Xianquan's multiplex qPCR kit targets the detection of Vibrio parahaemolyticus, Staphylococcus aureus and Listeria monocytogenes; He Peiyan's five-qPCR kit targets the detection of Staphylococcus aureus, Listeria monocytogenes, Salmonella, Vibrio parahaemolyticus and Shigella; Shanghai Cebio's seven-qPCR kit 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 Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus in aquatic products. Therefore, from the application point of view, it is of great significance to develop a rapid, accurate, simple and specific four-qPCR diagnostic method to simultaneously identify and detect Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus.

[0004] The four-qPCR detection method can simultaneously detect Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus in aquatic products. However, considering that four-qPCR needs to add four pairs of primers and four probes in one system, in order to ensure that primers and probes do not affect each other, primers and probes need to be highly specific to avoid binding or non-specific amplification between primers and probes. In addition, considering that the nucleic acid in the template is more complex, and the annealing temperature required by different primers may be different. Therefore, it is of great research and practical value to establish a four-qPCR detection method with Taqman probe, but it is quite difficult to achieve. SUMMARY

[0005] The present application aims at overcoming at least one deficiency of the prior art, and provides a quadruple Taqman qPCR reagent for detecting foodborne pathogenic bacteria in aquatic products.

[0006] The technical scheme adopted by the present application is:

[0007] In a first aspect, the present application provides a quadruple Taqman qPCR reagent for detecting foodborne pathogenic bacteria in aquatic products, the foodborne pathogenic bacteria being Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus; the reagent comprising specific primers and Taqman probes corresponding to the four foodborne pathogenic bacteria;

[0008] The primer sequence and Taqman probe sequence 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 the sequence shown in SEQ ID NO. 2;

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

[0012] The primer sequence and Taqman probe sequence 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 sequence and Taqman probe sequence for detecting Shigella are: upstream primer Sc-F, downstream primer Sc-R, Taqman probe Sc-P;

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

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

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

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

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

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

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

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

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

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

[0027] In some examples, different fluorescent-quenching groups are labeled on probes of different strains.

[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 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 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 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 BHQ2.

[0032] In a second aspect, the present application provides a quadruple Taqman qPCR kit for detecting foodborne pathogenic bacteria in aquatic products, which comprises the quadruple Taqman qPCR detection reagent of the first aspect.

[0033] In some examples, the foodborne pathogenic bacteria 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 of Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus.

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

[0037] In some examples, the lysis solution comprises 4.9-5.1 mmol / L tris (hydroxymethyl aminomethane) and 0.019-0.121% sodium dodecyl sulfate solution.

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

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

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

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

[0042] The present application has the following advantages:

[0043] 1. Since there is currently no effective method for simultaneously detecting foodborne pathogens in aquatic products, the present application designs a primer and probe set for detecting four foodborne pathogens in aquatic products by using multiplex qPCR technology according to the characteristics of important foodborne pathogens in aquatic products, including the upstream and downstream primers and probes for Vibrio parahaemolyticus, the upstream and downstream primers and probes for Vibrio vulnificus, the upstream and downstream primers and probes for Shigella, and the upstream and downstream primers and probes for Staphylococcus aureus, which correspond to SEQ ID NO 1, 2 and 3, SEQ ID NO 4, 5 and 6, SEQ ID NO 7, 8 and 9, and SEQ ID NO 10, 11 and 12 in the sequence listing, respectively. This multiplex detection method improves the detection efficiency and reduces the experimental cost.

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

[0045] 3. The four-fold Taqman probe qPCR detection method established by the application has high sensitivity, and the detection sensitivity of Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus can reach 2.0x10 2 CFU / mL, and the results have excellent repeatability. In addition, compared with the traditional biochemical identification relying on pure culture, the detection method can complete the whole process of sample detection (including the pre-inoculation process of the sample) within 8-11h, wherein the qPCR reaction time is 1h, and the manual operation time is 1h.

[0046] 4. In summary, the application provides a four-fold Taqman qPCR kit and a detection method thereof, the kit has the characteristics of high efficiency, accuracy, sensitivity and rapidness, and provides an accurate, rapid, efficient and economic means for the detection of the above-mentioned four foodborne pathogens in the quality and safety detection of aquatic products and their processed products, which has important significance for ensuring the edible safety of aquatic products in China. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 is a single qPCR method detection amplification curve diagram of Vibrio parahaemolyticus.

[0048] Figure 2 is a single qPCR method detection amplification curve diagram of Vibrio vulnificus.

[0049] Figure 3 is a single qPCR method detection amplification curve diagram of Shigella.

[0050] Figure 4 is a single qPCR method detection amplification curve diagram of Staphylococcus aureus.

[0051] Figure 5 is the sensitivity test result of the four-fold Taqman qPCR reaction system for detecting a single pathogen Vibrio parahaemolyticus.

[0052] Figure 6 is the sensitivity test result of the four-fold Taqman qPCR reaction system for detecting a single pathogen Vibrio vulnificus.

[0053] Figure 7 is the sensitivity test result of the four-fold Taqman qPCR reaction system for detecting a single pathogen Shigella.

[0054] Figure 8 is the sensitivity test result of the four-fold Taqman qPCR reaction system for detecting a single pathogen Staphylococcus aureus.

[0055] Figure 9The standard curve is the corresponding sensitivity test curve for detecting a single pathogen using a quadruple Taqman qPCR reaction system.

[0056] Figure 10 An example of multiplex qPCR detection using a mixture of four Cronobacter positive samples.

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

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

[0059] Figure 13 The amplification curve of Shigella in a sensitivity test of a quadruple Taqman qPCR reaction system for simultaneous detection of four pathogens is shown.

[0060] Figure 14 The amplification curve of Staphylococcus aureus in a sensitivity test of a quadruple Taqman qPCR reaction system for simultaneous detection of four pathogens is shown.

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

[0062] Figure 16 Figure 1 shows the anti-interference ability of the quadruple Taqman qPCR reaction system. Figure 2 shows the actual amplification curves of the four target bacteria after the addition of high-density interfering bacteria, and Figure 3 shows the Ct value line graph corresponding to Figure 4. Detailed Implementation

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

[0064] Example 1

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

[0066] Table 1. Specific qPCR primers and corresponding Taqman probe sequences for four foodborne pathogens.

[0067]

[0068]

[0069] Example 2

[0070] The quadruple Taqman qPCR detection method based on the detection reagent described in Example 1 is as follows:

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

[0072] S1) After picking suspicious colonies and culturing them continuously for 14 hours, take 1 mL of the enrichment broth into a 1.5 mL sterile centrifuge tube, centrifuge at 6000 r / min for 5 min, and completely remove the supernatant.

[0073] S2) Add 30 μL of lysis buffer prepared with 5 mmol / L sodium dodecyl sulfate (SDS) and 0.02% (m / v) tris-base. Dissolve the precipitate at the bottom of the tube completely with the lysis buffer, gently tap the tube wall to remove bubbles, and heat at 99°C for 10 min.

[0074] Centrifuge at 12000 rpm for 15 min (S3). The supernatant is the DNA sample to be tested. Place on ice and centrifuge again if not used for an extended period. Store all obtained DNA samples at -20℃ for later use.

[0075] (2) Four sets of qPCR primers and their corresponding probes were used for detection. The DNA obtained in (1) was used as a template. The primers and probes described in Example 1 were used for qPCR amplification and fluorescence signals were collected.

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

[0077] The qPCR amplification reaction program was as follows: the fluorescence channel was set to temperature control, and the program was set to 94℃ for 60s; 40 cycles included 95℃ for 20s and 59℃ for 30s; the fluorescence channels of the qPCR instrument were set as follows: Channel 1: FAM; Channel 2: Cy5; Channel 3: VIC; Channel 4: ROX; the qPCR instrument automatically collected fluorescence signals in the set fluorescence channels 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 >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 reagents described in Example 1 and the detection method described in Example 2 are as follows:

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

[0082] First, using the extracted genomic DNA of the target strain as a positive control template, and DEPC water as a blank control, with the four target pathogenic bacteria serving as negative controls for each other, singlet qPCR reactions were performed using the corresponding primers and probes to preliminarily demonstrate the specificity of the designed primer-probe set. The results are as follows: Figures 1-4 As shown, the primer-probe set designed for Vibrio parahaemolyticus, Vibrio vulnificus, Shigella, and Staphylococcus aureus can distinguish non-target bacteria with high specificity. 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 (setting concentration gradients of 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, and 0.5 μmol / L) were selected for single-factor optimization experiments. Taking into account the cycle threshold (Ct) and fluorescence signal value (ΔRn) of the amplification curve, the final singlet TaqMan qPCR reaction system for the four pathogenic bacteria was determined. Then, using the extracted genomic DNA of the target strains as a positive control template, orthogonal experiments were conducted with appropriate primer concentrations to optimize the multiplex qPCR reaction system based on the singlet qPCR method. The optimal conditions for the multiplex reaction were selected based on the Ct value, fluorescence intensity change, and sensitivity of the amplification curve, allowing the simultaneous detection of the four target foodborne pathogens in a single-tube system.

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

[0085] Table 2. Optimal selection of two multiplex qPCR reaction systems and comparison of mean Ct values.

[0086]

[0087] Note: The inoculation density for all four target bacteria was 2 × 10⁴. 7 CFU / mL

[0088] 2. Specificity validation of the multiplex qPCR system

[0089] To verify the specificity of the multiplex qPCR kit detection system constructed in this invention, genomic DNA of the target strain and other common non-target bacteria was extracted as templates based on the method described in Example 2. A 25 μL detection system was established according to the optimization results of Example 2 for multiplex qPCR detection, with each template subjected to three replicate reactions. A total of 105 strains were verified, including 53 target strains and 52 common non-target bacteria. The target strains were: 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 specificity verification results of the multiplex qPCR kit are shown in Table 3. Of the total 105 bacterial strains (including the target bacteria and other common species), only Vibrio parahaemolyticus, Vibrio vulnificus, Shigella, and Staphylococcus aureus showed positive amplification results in their corresponding fluorescence channels. This result demonstrates the high specificity of the multiplex qPCR kit detection system constructed in this invention.

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

[0092]

[0093]

[0094]

[0095] Note: "+" indicates a positive result, and "-" indicates a negative result.

[0096] Example 4: Quadruple Taqman qPCR Sensitivity Assay

[0097] Vibrio parahaemolyticus (ATCC 17802), Vibrio vulnificus (ATCC 27562), Shigella (CMCC(B)51105), and Staphylococcus aureus (ATCC 25923) were cultured overnight, and their bacterial densities were quantified using the plate count method. The quantitative results showed that the initial bacterial densities of Vibrio parahaemolyticus, Vibrio vulnificus, Shigella, and Staphylococcus aureus were 6.0 × 10⁻⁶. 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 culture and adjust the bacterial density of each culture to 2.0 × 10⁻⁶ by adding sterile physiological saline. 8 After reaching CFU / mL, seven 10-fold serial dilutions were performed (dilution factors: 0, -1, -2, -3, -4, -5, -6, -7). Subsequently, 1 mL of each bacterial genomic DNA was extracted from each serial dilution using the method described in Example 2. Multiplex qPCR amplification and detection were then performed based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2 to investigate the sensitivity of the detection method. Each reaction was repeated three times.

[0099] 2. Co-infection experiment: A certain volume of each target bacterium's original bacterial suspension and sterile physiological saline were taken and mixed to normalize the bacterial suspension density of each target bacterium in the mixture to 2.0 × 10⁻⁶.8 CFU / mL. This normalized mixture was then serially diluted 10-fold in seven different gradients. Subsequently, 1 mL of each of these gradient dilutions was taken to obtain genomic DNA according to the method described in Example 2, 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 to investigate the sensitivity of the detection method. Each reaction was repeated three times.

[0100] The results showed that this kit was effective at inoculation densities of 2.0 × 10⁻⁶. 7 Up to 2.0×10 2 Genomic DNA of Vibrio parahaemolyticus, Vibrio vulnificus, and Staphylococcus aureus at CFU / mL (6 gradients) and at an inoculation density of 2.0 × 10⁻⁶. 7 Up to 2.0×10 1 When Shigella genomic DNA was detected at CFU / mL, a good linear relationship (R0) was observed. 2 >0.99); the lowest detection density of Vibrio parahaemolyticus, Vibrio vulnificus, and Staphylococcus aureus can be as low as 2.0 × 10⁻⁶. 2 The lowest detectable density of Shigella can be as low as 2.0 × 10⁻⁶ CFU / mL. 1 CFU / mL. Standard curves for single and multiple pathogen detection were constructed based on the corresponding mean Ct values. For single pathogen detection, the slopes of the standard curves for Vibrio parahaemolyticus, Vibrio vulnificus, Shigella, and Staphylococcus aureus were -3.414, -3.387, -3.099, and -3.304, respectively, with corresponding amplification efficiencies of 96%, 97%, 110%, and 101%. For multiple pathogen detection, the slopes of the standard curves for Vibrio parahaemolyticus, Vibrio vulnificus, Shigella, and Staphylococcus aureus were -3.313, -3.439, -3.435, and -3.242, respectively, with corresponding amplification efficiencies of 100%, 95%, 95%, and 103%. The amplification efficiencies meet the threshold range (80%-120%) set for multiplex qPCR, indicating that this quadruple Taqman qPCR method has good amplification performance. This kit also demonstrates its ability to detect both single-infected and multi-infected samples. Detailed results are shown in Table 4 and... Figures 5-9 (Single pathogen infection) and Table 5 and Figures 10-13 (Multi-pathogen infection).

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

[0102]

[0103] Note: "–" indicates a negative result, and the same applies 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 from Example 4 as the input DNA template, a repeatability experiment (repeated 8 times) was conducted to test the "co-infection sensitivity" in Example 4, based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2. The stability of the method was assessed using the coefficient of variation (CV). Coefficient of variation = standard deviation / mean.

[0108] The results are shown in Table 6. When this quadruple qPCR was used to detect the normalized concentration gradient premix, the CV (%) value between the eight replicates of each detection gradient was basically less than 2%, and very few were between 2% and 3%, which met the precision threshold requirement (<15%) of the in vitro diagnostic kit, 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] Based on the characteristics of aquatic products, five foodborne pathogens—Salmonella typhimurium (ATCC 14028), Listeria monocytogenes (ATCC 19115), Escherichia coli O157:H7 (NCTC 12900), Aeromonas hydrophila (CMCC(B)10506), and Vibrio alginolyticus (ATCC 33787)—were selected as interfering bacteria. The selected five interfering bacteria and four target bacteria—Vibrio parahaemolyticus (ATCC 17802), Vibrio vulnificus (ATCC 27562), Shigella (CMCC(B)51105), and Staphylococcus aureus (ATCC 25923)—were cultured overnight, and their bacterial density was quantified using the plate count method. Quantitative results showed that the original bacterial cultures of Salmonella, Listeria monocytogenes, Escherichia coli O157:H7, Aeromonas hydrophila, and Vibrio alginolyticus had densities of 3.64 × 10⁻⁶. 8 2.58×10 9 1.30×10 8 3.21×10 8 4.08×10 8 The initial bacterial suspension densities of Vibrio parahaemolyticus, Vibrio vulnificus, Shigella, and Staphylococcus aureus were 2.13 × 10⁻⁶ CFU / mL, respectively. 8 3.50×10 8 8.75×10 8 8.82×10 8 CFU / mL. Then, the target bacterial suspension was serially diluted 10-fold in three gradients (dilution factors: 0, -1, -2, -3), and 2 mL of each of the four target bacterial dilutions (dilution factor -3) was mixed. Subsequently, 1 mL of each of the five interfering bacteria stock solutions (dilution factor -1), namely *Escherichia coli* O157:H7, *Salmonella typhimurium*, *Vibrio alginolyticus*, *Aeromonas hydrophila*, and *Listeria monocytogenes*, was added to 1 mL of the prepared target bacterial dilution mixture and mixed thoroughly. 1 mL of this mixture was then used to extract DNA according to the method described in Example 2, 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. The results are shown in Table 7. Figure 14 As shown, high-density interfering bacteria (10 8 The addition of CFU / mL did not significantly interfere with the detection of the target bacteria, and the Ct values ​​remained basically the same.

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

[0117]

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

[0119] Example 7: Artificially Contaminated Sample Test

[0120] (1) Preparation of bacterial serial dilution: Vibrio parahaemolyticus (ATCC 17802), Vibrio vulnificus (ATCC27562), Shigella (ATCC 50115) and Staphylococcus aureus (ATCC 25923) were cultured overnight, and the bacterial density of these four pathogens was quantified by plate counting method, and 10-fold serial dilutions were performed in six gradients (dilution factors: 0, -1, -2, -3, -4, -5, -6).

[0121] (2) Sample preparation: Under aseptic conditions, 25g of sterilized shrimp meat was added to 225ml of 3.5% sterile NaCl solution. After homogenization, 9ml of the homogenate was added to 90ml of the corresponding culture medium: 3% NaCl alkaline peptone water (Vibrio parahaemolyticus), PNCC (Vibrio vulnificus), GN (Shigella), and 7.5% NaCl broth (Staphylococcus aureus). 1mL of each gradient of the bacterial suspension prepared in (1) was added to the corresponding enrichment broth. The last four gradients (dilution factors: -3, -4, -5, -6) were cultured and enriched in a shaker at 37℃ (155r / min) for 3, 6, and 9h. After completion, 1mL of DNA was extracted from each gradient of each target bacterium as a template. Based on the optimal multiplex qPCR reaction system explored in Example 3 and the reaction procedure described in Example 2, multiplex qPCR amplification and detection were performed.

[0122] When the dilution factor is -6, the bacterial concentration in the simulated sample is approximately 1–10 CFU / mL, equivalent to a bacterial count of 1–10 CFU / g in the sample. Amplification results for the artificially contaminated samples are detailed in Table 8. Without enrichment culture, the positive detection limit for Vibrio parahaemolyticus and Vibrio vulnificus in the simulated samples is 10⁻⁶. 2 CFU / mL (dilution factor -4), the limit of detection for Shigella in the simulated sample is 10. 1 CFU / mL (dilution factor -5), the limit of detection for Staphylococcus aureus in the simulated sample is 10. 3 (CFU / mL dilution factor: -3); after 6 hours of enrichment, the positive detection limits for Vibrio parahaemolyticus, Vibrio vulnificus, Shigella, and Staphylococcus aureus in the simulated samples all reached 10. 0CFU / mL (dilution factor -6). However, for Shigella and Staphylococcus aureus, an enrichment time of up to 9 hours is preferable. Overall, compared to the traditional method which relies on a detection cycle of at least 18 hours, this method can complete the detection results within 8–11 hours, including 6–9 hours of enrichment, 1 hour of qPCR, and 1 hour of manual operation time, demonstrating strong time and cost-effectiveness.

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

[0124]

[0125]

[0126]

[0127]

[0128] Example 8: Clinical Trial Testing of Aquatic Products Using Multiplex qPCR

[0129] The established multiplex qPCR method was used to test 14 aquatic product samples purchased from local supermarkets and farmers' markets, including salmon (n=2), tilapia (n=3), prawns (n=1), sand shrimp (n=1), tiger prawns (n=1), kelp (n=1), cuttlefish balls (n=1), squid (n=1), squid tentacles (n=1), oysters (n=1), and shrimp meat (n=1). Specific method: 25g of each sample was weighed and added to the corresponding enrichment broth described in Example 7. After homogenization, the samples were incubated at 37℃ for 6–9 h. 1 mL of DNA was extracted from each sample and used as an amplification template. 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. Meanwhile, 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 respectively, and the test results of the two methods were compared.

[0130] The results showed that, using this multiplex qPCR kit, 10 out of 14 aquatic product samples tested 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 samples of Vibrio parahaemolyticus, 6 for Vibrio vulnificus, 4 for Staphylococcus aureus, and 0 for Shigella. In general, the results of samples positive by traditional methods were consistent with those of qPCR, but the positive detection rate of foodborne pathogens using qPCR was slightly higher than that using traditional methods (Table 9). The inconsistency in the results can be attributed to the fact that traditional detection methods rely on obtaining pure bacterial cultures, which may lead to the omission of target bacteria during the purification process.

[0131] Table 9 Comparison of detection results of four target bacteria in 14 aquatic product samples by quadruple Taqman qPCR and national standard identification method.

[0132]

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

[0134] In summary, the above embodiments demonstrate 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 and repeatability, and high sensitivity. It is suitable for the 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 and should not be considered as a limitation on the specific implementation of the present invention. For those skilled in the art, simple deductions or substitutions without departing from the concept of the present invention are all within the protection scope of the present invention.

Claims

1. A quadruple Taqman qPCR reagent for detecting foodborne pathogenic bacteria in aquatic products, characterized in that, The foodborne pathogenic bacteria are Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus; the reagent comprises four specific primers and Taqman probes corresponding to the four foodborne pathogenic bacteria; The primer sequence and Taqman probe sequence 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 the sequence shown in SEQ ID NO. 2; The Vp-P has the sequence shown in SEQ ID NO. 3; The primer sequence and Taqman probe sequence 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 sequence and Taqman probe sequence for detecting Shigella are: upstream primer Sc-F, downstream primer Sc-R, Taqman probe Sc-P; The Sc-F has the sequence shown in SEQ ID NO. 7; The Sc-R has the sequence shown in SEQ ID NO. 8; The Sc-P has the sequence shown in SEQ ID NO. 9; The primer sequence and Taqman probe sequence 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 the sequence shown in SEQ ID NO. 10; The Sau-R has the sequence shown in SEQ ID NO. 11; The Sau-P has the sequence shown in SEQ ID NO. 12; The Sau-F1 has the sequence shown in SEQ ID NO. 13; The Sau-R1 has the sequence shown in SEQ ID NO. 14; The Sau-P1 has the sequence shown in SEQ ID NO. 15; The probes of different strains are labeled with different fluorescence-quenching groups.

2. The quadruplex Taqman qPCR reagent according to claim 1, 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 quenching 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 quenching 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 quenching 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 quenching group BHQ2.

3. A quadruple Taqman qPCR kit for detecting foodborne pathogenic bacteria in aquatic products, characterized in that, The quadruple Taqman qPCR kit comprises the quadruple Taqman qPCR detection reagent of claim 1 or 2.

4. The quadruplex Taqman qPCR kit according to claim 3, characterized in that, The foodborne pathogenic bacteria are Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus.

5. The quadruplex Taqman qPCR kit according to claim 3, characterized in that, The kit further comprises a negative control, a positive control and a lysis solution.

6. The quadruplex Taqman qPCR kit according to claim 5, characterized in that, The positive control is a mixture of genomic DNA of Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus.

7. The quadruplex Taqman qPCR kit according to claim 5, characterized in that, The negative control is DEPC water.

8. The quadruplex Taqman qPCR kit according to claim 5, characterized in that, The lysis solution is composed of 4.9-5.1 mmol / L tris (hydroxymethyl aminomethane) and 0.019-0.121% sodium dodecyl sulfate solution.

9. A method for detecting a quadruple Taqman qPCR detection reagent for detecting Vibrio parahaemolyticus, Vibrio vulnificus, Shigella and Staphylococcus aureus in aquatic products, characterized by, The method comprises the following steps: 1) extracting genomic DNA of the sample to be tested for standby; 2) adding the genomic DNA of the sample to be tested as a template into a quadruplex qPCR reaction system for amplification, wherein the reaction system comprises the quadruplex Taqman qPCR kit according to any one of claims 3-8; 3) setting a qPCR program and collecting fluorescence signals in the amplification process, and determining the results.

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