A multiplex PCR primer set and detection method for simultaneously detecting five foodborne pathogenic bacteria in tilapia

Abstract

This application discloses a multiplex PCR primer set and detection method for the simultaneous detection of five foodborne pathogens in tilapia. The multiplex PCR detection primers specifically target *Streptococcus agalactiae*. ef-tμ Genes, Aeromonas hydrophila ompA Genes, Salmonella invA Genes, Vibrio cholerae ompW Genes, E. coli phoA The primer pair composition for the gene is shown in Sequence Listing 2. Based on the above primer pairs, this invention optimized the multiplex PCR detection system and reaction conditions, established a five-fold PCR detection method, and evaluated the specificity, sensitivity, and detection of artificial simulated samples of the multiplex PCR system. The sensitivity was above 0.4 ng / μL, and the detection limit reached 10. 2 CFU / mL. The establishment of this method provides an important technical means for the rapid detection of common foodborne pathogens in tilapia, and also lays the foundation for the prevention and control of bacterial diseases in tilapia.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a multiplex PCR primer set and detection method for simultaneously detecting five foodborne pathogens in tilapia. Background Technology

[0002] Tilapia is one of my country's most important exported aquatic products. As the world's largest exporter of tilapia, my country accounts for more than 50% of the global tilapia production annually. Tilapia product safety plays a crucial role in my country's food safety strategy. Foodborne pathogens have become a key issue threatening tilapia safety and impacting human health. Accurate and rapid detection of foodborne pathogens in tilapia is essential for controlling foodborne disease outbreaks. Currently, the detection of foodborne pathogens in tilapia mainly targets Streptococcus agalactiae, with relatively less detection of other pathogens. However, research indicates that tilapia can become contaminated during processing, packaging, transportation, storage, and sales due to contact with various environmental pathogens. The investigation by Li Xiaozheng et al. on foodborne pathogen contamination in tilapia in Guangxi Province showed that various foodborne pathogens, including Streptococcus agalactiae, Aeromonas hydrophila, Salmonella, and Escherichia coli, were detected in both farmed and commercially available tilapia samples. Li Laihao et al.'s analysis of tilapia samples from different farms in Guangdong Province showed high detection rates of Escherichia coli, Vibrio cholerae, and Aeromonas hydrophila, followed by Enterobacter akazakii and Vibrio parahaemolyticus. Therefore, there is an urgent need to establish a rapid, sensitive, and specific pathogen detection technology to prevent and control foodborne diseases in tilapia.

[0003] Currently, the detection of pathogenic bacteria in aquatic animals typically employs conventional techniques such as culture isolation and biochemical identification. This method is time-consuming, complex, and limited to a single pathogen, making it difficult to meet the needs of rapid testing in modern food safety. Although some researchers have developed multiplex PCR methods capable of simultaneously detecting multiple pathogens—for example, Hu Zongyun et al. established a multiplex PCR method for simultaneously detecting three pathogenic bacteria in freshwater fish; Jiang Wei et al. established a triplex PCR method for detecting Vibrio cholerae, Vibrio parahaemolyticus, and Listeria monocytogenes in aquatic products; and Wang Lu et al. established a dual PCR method for rapidly detecting Aeromonas hydrophila based on cellular excitotoxin genes and aerolysin structural genes—these methods can only detect 1-3 pathogens, resulting in a narrow detection range, low efficiency, and unsuitability for detecting multiple foodborne pathogens in tilapia. Summary of the Invention

[0004] To overcome the problems in existing technologies and achieve rapid, sensitive, and high-throughput detection of common foodborne pathogens in tilapia, this study established a five-fold PCR detection system using the *Streptococcus agalactiae* ef-tμ gene, *Aeromonas hydrophila* ompA gene, *Salmonella invA* gene, *Vibrio cholerae* ompW gene, and *Escherichia coli* phoA gene as target genes. This provides an important technical means for the rapid and efficient detection of common foodborne pathogens in tilapia.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a multiplex PCR primer set for simultaneous detection of five foodborne pathogens in tilapia. The five bacteria that can be detected simultaneously using the primer set are Streptococcus agalactiae, Aeromonas hydrophila, Vibrio cholerae, Escherichia coli, and Salmonella. The primer set is designed targeting the ef-tμ gene of Streptococcus agalactiae, the ompA gene of Aeromonas hydrophila, the invA gene of Salmonella, the ompW gene of Vibrio cholerae, and the phoA gene of Escherichia coli.

[0006] Preferably, the primer set includes upstream primers with sequences as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, and downstream primers with sequences as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10.

[0007] Preferably, in the primer set,

[0008] Sequences SEQ ID NO: 1 and SEQ ID NO: 2 target the ef-tμ gene of Streptococcus agalactiae;

[0009] The sequences SEQ ID NO: 3 and SEQ ID NO: 4 target the ompA gene of Aeromonas hydrophila;

[0010] Or the sequences SEQ ID NO: 5 and SEQ ID NO: 6 target the Salmonella invA gene;

[0011] Or the sequences SEQ ID NO: 7 and SEQ ID NO: 8 target the ompW gene of Vibrio cholerae;

[0012] The sequences SEQ ID NO: 9 and SEQ ID NO: 10 target the phoA gene in Escherichia coli.

[0013] As another aspect of the present invention, the present invention provides a multiplex PCR detection method for simultaneously detecting five foodborne pathogens in tilapia, which includes the following steps:

[0014] S1 extracts total DNA from the sample to be tested;

[0015] S2 uses extracted genomic DNA as a template and performs multiplex PCR amplification using a primer set;

[0016] Determination of S3 amplification products: PCR products were analyzed by agarose gel electrophoresis;

[0017] If the PCR product fragment size contains one or more of the following values: 150bp, 349bp, 474bp, 588bp, and 830bp, it indicates that the sample contains one or more of the following bacteria: Streptococcus agalactiae, Aeromonas hydrophila, Escherichia coli, Vibrio cholerae, and Salmonella.

[0018] Preferably, the multiplex PCR amplification reaction system in step S2 is as follows: 5 μL template, the concentration of each primer pair in the primer set is 0.8-1.4 μmol / L, 20 μL 2×Mix, and ddH2O to make up to 40 μL;

[0019] Preferably, the multiplex PCR amplification reaction conditions in step S2 are as follows: pre-denaturation at 95℃ for 3 min, denaturation at 95℃ for 30 s, annealing at 50.9–57.1℃ for 30 s, extension at 72℃ for 30 s, running for 32–36 cycles, full extension at 72℃ for 10 min, and storage of the amplification product at 4℃.

[0020] Preferably, the multiplex PCR amplification reaction conditions in step S2 are as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 52.9℃ annealing for 30 s, 72℃ extension for 30 s, run for 32 cycles, 72℃ full extension for 10 min, and store the amplification product at 4℃.

[0021] The beneficial effects of this invention are:

[0022] The multiplex PCR method established in this study can rapidly detect five common foodborne pathogens in tilapia, including Streptococcus agalactiae, Aeromonas hydrophila, Vibrio cholerae, Escherichia coli, and Salmonella, with good specificity and sensitivity. The entire process, including total DNA extraction, PCR amplification, and electrophoretic analysis, can be completed within 16 hours, meeting the needs of large-scale testing of aquatic products and showing broad application prospects. Attached Figure Description

[0023] Figure 1 These are the results of five-fold PCR amplification with different primer concentrations: M: DL2000 Maker; 1-8: 1.4, 1.2, 1.0, 0.1, 0.8, 0.6, 0.4, 0.2 μmol / L.

[0024] Figure 2 These are the results of five-fold PCR amplification at different annealing temperatures. M: DL2000 Maker; 1-6: annealing temperatures were 50.9℃, 52.9℃, 55.0℃, 57.1℃, 59.1℃, and 61.1℃, respectively.

[0025] Figure 3 These are the results of five-fold PCR amplification at different annealing times. M: DL2000 Maker; 1-6: annealing times are 15s, 20s, 25s, 30s, 35s, and 40s, respectively.

[0026] Figure 4 These are the results of five-fold PCR amplification with different number of cycles. M: DL2000 Maker; 1-6: cycle numbers are 26, 28, 30, 32, 34, and 36, respectively.

[0027] Figure 5 These are the results of specific tests for five PCR reaction systems: M: DL2000 Maker; 1: 5 pairs of specific primers and genomic DNA of 5 target strains, as a positive control group; 2: 5 pairs of specific primers and genomic DNA of 5 target strains and 5 non-target strains; 3: 5 pairs of specific primers and genomic DNA of 5 non-target strains, as a negative control group.

[0028] Figure 6 These are the results of sensitivity tests for five PCR reaction systems. M: DL2000 Maker; 1-6: bacterial concentrations of 10... 6 10 5 10 4 10 3 10 2 10 1 CFU / mL;

[0029] Figure 7The results are from multiplex PCR detection of artificially simulated samples;

[0030] Figure 8 The PCR results for other primers targeting the five target strains are shown in Figure A, where Aeromonas hydrophila and Salmonella did not amplify a specific band; Figure B, where Streptococcus agalactiae did not amplify a specific band; Figure C, where Vibrio cholerae and Escherichia coli did not amplify a specific band; and Figure D, where Vibrio cholerae did not amplify a specific band. Detailed Implementation

[0031] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be described in further detail below with reference to the accompanying drawings. However, the implementation of the present invention is not limited thereto. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0032] The strains used in the following examples are as follows:

[0033] Table 1 Test strains

[0034]

[0035] 1.Pang MB, Jiang JW, Xie

[0036] 2. Liu G, Zhang W, Lu C. Complete genome sequence of Streptococcus agalactiaeGD201008-001, isolated in China from tilapia with meningoencephalitis. Journal of bacteriology. 2012; 194(23):6653.

[0037] Example 1: Extraction of bacterial genomic DNA

[0038] Bacterial genomic DNA was extracted using a bacterial genome extraction kit. DNA concentration was determined using a Nano Drop instrument and then stored at -20°C for later use.

[0039] Example 2: Primer Design and Synthesis

[0040] The published whole genome sequences of Streptococcus agalactiae, Aeromonas hydrophila, Vibrio cholerae, Escherichia coli, and Salmonella were downloaded from the GenBanK database. Conserved target genes were screened through comparative genomics analysis. Primers were designed using Primer Premier 5.0 software based on the conserved gene sequences and synthesized by Suzhou Genewiz Biotechnology Co., Ltd. The primer sequences and related information are shown in Table 2.

[0041] Table 2 Primer Information

[0042]

[0043] Example 3: Establishment and optimization of multiplex PCR reaction system

[0044] A preliminary multiplex PCR reaction system was established: 20 μL of 2×Taq Mix, 1 μL of each primer (final concentration 0.2 μmol / L), 1 μL of DNA from each of the five target strains as template, and ddH2O added to a final volume of 40 μL. The reaction program was: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 15 s, 30 cycles; final extension at 72℃ for 5 min.

[0045] (1) Primer concentration optimization

[0046] Multiplex PCR amplification was performed using primer pairs at concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.4 μmol / L. The results are as follows: Figure 1 The optimal primer concentrations for Salmonella, Vibrio cholerae, and Escherichia coli are 0.8 μmol / L, while the optimal primer concentrations for Streptococcus agalactiae and Aeromonas hydrophila are 1.2 μmol / L.

[0047] (2) Annealing temperature optimization

[0048] Annealing temperatures of 50.9℃, 52.9℃, 55.0℃, 57.1℃, 59.1℃, and 61.1℃ were set for multiplex PCR amplification. The results showed that the amplified bands were clearer when the annealing temperature was between 50.9℃ and 57.1℃. However, when the annealing temperature exceeded 57.1℃, the target bands for *Streptococcus agalactiae*, *Aeromonas hydrophila*, and *Vibrio cholerae* were less distinct. Figure 2 Based on the clarity of each stripe, the optimal annealing temperature was determined to be 52.9℃.

[0049] (3) Annealing time optimization

[0050] Annealing times were set to 15s, 20s, 25s, 30s, 35s, and 40s for multiplex PCR amplification. The results are as follows: Figure 3 As shown, when the annealing time is 30s, the bands of each target are clearer, so the optimal annealing time is 30s.

[0051] (4) Loop number optimization

[0052] Multiplex PCR amplification was performed with amplification cycle numbers of 26, 28, 30, 32, 34, and 36, respectively. Figure 4 As shown, when the number of cycles is 32, 34, and 36, the amplification effect is good and there is no significant difference. Considering the need for rapid detection, the number of cycles is determined to be 32.

[0053] Example 4: Multiplex PCR Specificity Assay

[0054] Genomic DNA from five target bacteria and five non-target bacteria (Vibrio parahaemolyticus, Staphylococcus aureus, Vibrio vulnificus, Listeria monocytogenes, and Cronobacter sakazakii) was used in PCR reactions with five pairs of specific primers. The combinations were as follows: Group 1: five pairs of specific primers with genomic DNA from the five target strains (positive control); Group 2: five pairs of specific primers with genomic DNA from the five target strains and five non-target strains; Group 3: five pairs of specific primers with genomic DNA from the five non-target strains (negative control). Amplification was performed using an optimized multiplex PCR system, and the amplified products were analyzed by 1.5% agarose gel electrophoresis. The results showed that the expected fragment could be amplified using a mixed genome of Streptococcus agalactiae, Aeromonas hydrophila, Vibrio cholerae, Escherichia coli, and Salmonella as a template. However, no nucleic acid band amplification was observed when using a mixed genome of the five non-target bacteria (Vibrio parahaemolyticus, Staphylococcus aureus, Vibrio vulnificus, Listeria monocytogenes, and Cronobacter sakazakii) as a template. Figure 5 This indicates that the constructed multiplex PCR method has good specificity.

[0055] Example 5: Multiplex PCR Sensitivity Assay

[0056] The genomic mixtures of five target bacteria were serially diluted 10-fold to determine their sensitivity for multiplex PCR. The results showed that the sensitivity for *Streptococcus agalactiae*, *Aeromonas hydrophila*, *Salmonella*, *Vibrio cholerae*, and *Escherichia coli* was all above 0.4 ng / μL. Figure 6 ).

[0057] Example 6: Multiplex PCR detection of artificially simulated samples

[0058] After overnight incubation of Streptococcus agalactiae, Aeromonas hydrophila, Vibrio cholerae, Escherichia coli, and Salmonella, the bacterial concentration was adjusted to 5 × 10⁻⁶. 7CFU / mL was used for serial dilution with 10-fold serial dilutions. 200 μL of bacterial culture for each dilution was mixed into one EP tube. 5 g of tilapia gill tissue was added to 100 μL of each of the different concentration gradients of the mixed bacterial culture. The tissue was aseptically minced and ground, and the enrichment solution was brought to 10 mL. The mixture was incubated at 37°C for 12 h. Genomic DNA was extracted from 0.5 mL of each culture using a kit method. Amplification was performed according to the optimized multiplex PCR system, and the results were analyzed by 1.5% agarose gel electrophoresis. The results showed that the sensitivity for Streptococcus agalactiae, Aeromonas hydrophila, Salmonella, Vibrio cholerae, and Escherichia coli was 10. 2 CFU / mL Figure 7 This indicates that the multiplex PCR detection method established in this study can be applied to subsequent clinical sample testing.

[0059] Comparative Example 1

[0060] Primer selection: In the early stages of experimentation, different primers were tried for each bacterium to establish a multiplex PCR system, but the results were not ideal. Figure 8 ).

[0061] Multiplex PCR reaction system: 20 μL of 2×Taq Mix, 1 μL of each primer (final concentration 0.2 μmol / L), 1 μL of DNA from each of the 5 target strains as template, and ddH2O to a final volume of 20 μL. Reaction program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 15 s, 30 cycles; final extension at 72℃ for 5 min.

[0062] Figure 8 Primer information used in A

[0063]

[0064]

[0065] Figure 8 Information on primers used in B

[0066]

[0067] Figure 8 Primer information used in C

[0068]

[0069] Figure 8 Primer information used in D

[0070]

[0071] The possible reasons for the above experimental results are as follows: (1) primer dimers may form between different primers; (2) the optimal annealing temperature is inconsistent; (3) the primers have poor specificity.

[0072] The key and challenge in establishing multiplex PCR lies in primer design. It's crucial to consider the specificity of each primer pair as well as interference between primers; to minimize differences in annealing temperatures among primers; and to ensure the amplified fragments are easily distinguishable. Because the microenvironment within organisms is highly complex, designed primers can exhibit significant differences in performance. This experiment screened out the primers with the best amplification effect by experimenting with different primer combinations.

[0073] This study used the template dilution method to determine the limit of detection for multiplex PCR. However, some studies use serial dilution of bacterial culture before extracting the genome as a template for detection. The amount of DNA extracted by this method is affected by various factors such as kit efficiency and individual operation, which may result in a large error in the limit of detection, making it unsuitable for determining the limit of detection.

[0074] In summary, the multiplex PCR detection method established in this study, which can simultaneously detect five foodborne pathogens in aquatic products—Streptococcus agalactiae, Aeromonas hydrophila, Salmonella, Vibrio cholerae, and Escherichia coli—exhibits good specificity and sensitivity, and has a short detection time. It can provide a technical means for the rapid diagnosis of common foodborne pathogens in tilapia.

[0075] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the principles of this application should be included within the protection scope of this application.

Claims

1. A multiplex PCR primer set for simultaneous detection of five foodborne pathogens in tilapia, characterized in that: The primer set can simultaneously detect five bacteria: Streptococcus agalactiae, Aeromonas hydrophila, Vibrio cholerae, Escherichia coli, and Salmonella. The primer set is designed for the ef-tμ gene of Streptococcus agalactiae, the ompA gene of Aeromonas hydrophila, the invA gene of Salmonella, the ompW gene of Vibrio cholerae, and the phoA gene of Escherichia coli. The primer set consists of upstream primers with sequences as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 and SEQ ID NO: 9, and downstream primers with sequences as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10; In the primer set Sequences SEQ ID NO: 1 and SEQ ID NO: 2 target the ef-tμ gene of Streptococcus agalactiae; Sequences SEQ ID NO: 3 and SEQ ID NO: 4 target the ompA gene of Aeromonas hydrophila; Sequences SEQ ID NO: 5 and SEQ ID NO: 6 target the Salmonella invA gene; Sequences SEQ ID NO: 7 and SEQ ID NO: 8 target the ompW gene of Vibrio cholerae; Sequences SEQ ID NO: 9 and SEQ ID NO: 10 target the phoA gene in Escherichia coli. Multiplex PCR amplification using primer sets: if the PCR product fragments contain sizes of 150bp, 349bp, 474bp, 588bp, and 830bp, it indicates that the sample contains Streptococcus agalactiae, Aeromonas hydrophila, Escherichia coli, Vibrio cholerae, and Salmonella, respectively.

2. The multiplex PCR primer set for simultaneous detection of five foodborne pathogens in tilapia according to claim 1, characterized in that: Includes the following steps, S1 extracts total DNA from the sample to be tested; S2 uses extracted genomic DNA as a template and performs multiplex PCR amplification using a primer set; Determination of S3 amplification products: PCR products were analyzed by agarose gel electrophoresis; If the PCR product fragment sizes contain 150bp, 349bp, 474bp, 588bp, and 830bp, it indicates that the sample contains *Streptococcus agalactiae*, *Aeromonas hydrophila*, *Escherichia coli*, *Vibrio cholerae*, and *Salmonella*, respectively. The multiplex PCR amplification reaction system in step S2 is as follows: 5 μL template, primer concentrations of each primer pair in the primer set are 0.8~1.4 μmol / L, 20 μL 2×Mix, and ddH2O to make up to 40 μL. The multiplex PCR amplification reaction conditions in step S2 are as follows: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 52.9℃ annealing for 30 s, 72℃ extension for 30 s, run for 32 cycles, 72℃ extension for 10 min, and store the amplification product at 4℃.

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