Specific target, primer, detection method and application for detecting vibrio vulnificus

Abstract

The application discloses a specific target point for detecting Vibrio vulnificus, a primer, a detection method and application, and belongs to the technical field of food safety detection. The new specific target point for detecting Vibrio vulnificus is screened by establishing a local database and an online comparison method. A double digital PCR method is established by using the new target point and a traditional target point. The PCR primer probe is designed according to the self-screening target gene AOT11_08030 (vv08030) and the traditional target gene vvhA. In order to ensure the specificity and sensitivity of the method, the PCR amplification conditions and the reaction system are reasonably optimized. The experimental results show that the method has the characteristics of high specificity, high sensitivity and simple result determination. The detection process does not need to be opened, and the tedious electrophoresis operation is omitted. The detection time is shortened to 3 hours or less, and the method is suitable for rapid quantitative detection of Vibrio vulnificus in food.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing technology, specifically relating to a specific target, primers, detection method, and application for detecting Vibrio vulnificus. Background Technology

[0002] Vibrio vulnificus (Vv) is one of the most common pathogenic Vibrio bacteria, widely distributed in seawater and seafood such as shellfish (oysters). Infection can occur through consumption of raw or undercooked seafood, as well as through contact with wounds, especially in patients with liver disease and those with weakened immune systems. Many patients with Vibrio vulnificus infection require intensive care or amputation, and approximately 20% of infected patients die. Furthermore, Vibrio vulnificus is a significant contributor to necrotizing fasciitis, causing tissue death around open wounds, hence its nickname "flesh-eating bacteria." Currently, detection of Vibrio vulnificus primarily relies on the traditional culture and identification method of GB4789.44-2020. This standard, the first national food safety standard for Vibrio vulnificus testing in my country released in 2020, generally includes pre-enrichment, initial screening with conventional PCR, selective plate isolation, pure culture, and biochemical reactions, a process that typically takes 3–6 days.

[0003] With the rapid development and application of molecular biology, molecular detection techniques based on nucleic acid amplification have received widespread attention both domestically and internationally. These mainly include conventional PCR, quantitative real-time PCR (qPCR), and droplet digital PCR (ddPCR). The accuracy and specificity of these methods largely depend on the selection of target genes. Currently, commonly used targets for Vibrio vulnificus detection mainly focus on genes encoding DNA gyrase B subunits (gyrB), transmembrane transcription factor (toxR), cytolysin genes (vvhA), and metalloproteinase genes (met). However, in actual detection, the specificity of some of these genes is relatively low, leading to reduced accuracy and failing to fully leverage the advantages of molecular detection techniques, especially PCR. Currently, research on PCR detection targets for Vibrio vulnificus is limited; therefore, identifying novel and highly specific molecular targets is of great significance. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the first objective of this invention is as follows: 1)-6) the application of any gene fragment as a specific target in the detection of Vibrio vulnificus, wherein the application is not for the purpose of disease diagnosis:

[0005] 1)AOT11_08030; 2)AOT11_05055; 3)AOT11_09030; 4)AOT11_17375; 5)AOT11_17485; 6)AOT11_21945;

[0006] The nucleotide sequence of AOT11_08030 is shown in SEQ ID NO.1;

[0007] The nucleotide sequence of AOT11_05055 is shown in SEQ ID NO.2;

[0008] The nucleotide sequence of AOT11_09030 is shown in SEQ ID NO.3;

[0009] The nucleotide sequence of AOT11_17375 is shown in SEQ ID NO.4;

[0010] The nucleotide sequence of AOT11_17485 is shown in SEQ ID NO.5;

[0011] The nucleotide sequence of AOT11_21945 is shown in SEQ ID NO.6.

[0012] In one embodiment, the application includes the preparation of a detection reagent or kit for Vibrio vulnificus.

[0013] A second objective of this invention is to provide a detection composition for detecting Vibrio vulnificus, the composition comprising primer set A for the target AOT11_08030 (located on chromosome 1, accession number: CP012881.1) and primer set B for the conventional target vvhA (located on chromosome 2, accession number: CP012882.1).

[0014] In one embodiment, primer set A includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8, and a probe with a nucleotide sequence as shown in SEQ ID NO.9.

[0015] In one embodiment, primer set B includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO. 10, a downstream primer with a nucleotide sequence as shown in SEQ ID NO. 11, and a probe with a nucleotide sequence as shown in SEQ ID NO. 12.

[0016] In one embodiment, the 5' end of the probe is modified with a fluorescent reporter group, and the 3' end of the probe is modified with a quencher group.

[0017] In one embodiment, the reporter group is selected from, but not limited to, FAM, HEX, JOE, CY3, and CY5, and the quencher group is selected from, but not limited to, DABCYL, TAMRA, and BHQ.

[0018] In one embodiment, the 5' end of the probe of primer set A is modified with a fluorescent reporter group HEX, and the 5' end of the probe of primer set B is modified with a fluorescent reporter group FAM.

[0019] A third objective of this invention is to provide a kit for detecting Vibrio vulnificus, the kit comprising the above-described detection composition.

[0020] In one embodiment, the kit also contains a standard for Vibrio vulnificus.

[0021] The fourth objective of this invention is to provide a method for detecting Vibrio vulnificus for non-disease diagnostic purposes, comprising the following steps:

[0022] (1) Extract genomic DNA from the sample to be tested or directly dilute the sample to be tested;

[0023] (2) Use the above detection composition to perform qPCR or ddPCR.

[0024] In one embodiment, in step (2), the ddPCR reaction system is made by adding a detection composition, ddPCR reaction reagent and ultrapure water to a 4 μL sample of genomic DNA or a diluted sample of the test as a template.

[0025] In one embodiment, in step (2), the qPCR reaction system is made by adding a detection composition, qPCR reaction reagent and ultrapure water to a 5 μL sample of genomic DNA or a diluted sample of the test as a template.

[0026] In one embodiment, the ddPCR reaction system consists of 10 μL of ddPCR Supermix for Probes (NodUTP), 1.0 μL each of upstream and downstream primers (10 μmol / L) for vvhA and vv08030 genes, 0.5 μL of probe (10 μmol / L), 4 μL of DNA template, and finally water to a final volume of 20 μL.

[0027] In one implementation, the ddPCR amplification program is as follows: 95℃ for 10 min; 94℃ for 30 s; 60℃ for 1 min, 40 cycles; 98℃ for 10 min; and storage at 4℃ (the temperature rise and fall rate for all temperatures in the program is 2℃ / s). After amplification, the readings are taken using a QX200 Droplet Reader.

[0028] In one embodiment, the qPCR reaction system consists of 12.5 μL of Premix Ex Taq (Probe qPCR), 1.0 μL each of the upstream and downstream primers (10 μmol / L) for vvhA and vv08030 genes, 0.5 μL of probe (10 μmol / L), 5 μL of DNA template, and finally water to a final volume of 25 μL.

[0029] In one embodiment, the qPCR amplification program is 95℃ for 30s; 95℃ for 5s, 60℃ for 30s, for 40 cycles.

[0030] The present invention has the following beneficial effects:

[0031] (1) Specific detection targets for Vibrio vulnificus were obtained through bioinformatics methods such as the construction of a local database and genome alignment analysis: AOT11_08030, AOT11_05055, AOT11_09030, AOT11_1737, AOT11_17485, and AOT11_21945 were used in the detection of Vibrio vulnificus. The application was not for the purpose of disease diagnosis, but increased the detectable targets of Vibrio vulnificus. At the same time, the detection targets provided by this invention have good specificity. Among the 14 strains, only Vibrio vulnificus showed amplification signals. Other Vibrio species (Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio cholerae) and negative control bacteria (Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa, Bacillus cereus) showed no amplification.

[0032] (2) The detection method of the present invention can directly identify Vibrio vulnificus. It uses a combination of self-screening specific targets and traditional targets to detect Vibrio vulnificus, which has high specificity and reduces the occurrence of missed detections and false negatives.

[0033] (3) Compared with conventional PCR, the method for detecting Vibrio vulnificus provided by this invention does not require opening the sample container, reducing cross-contamination between samples, eliminating cumbersome downstream electrophoresis and other operations, and shortening the detection time. Compared with dye-based fluorescent PCR, there is no need to set a melting curve, and the use of fluorescently modified probes enhances the specificity of fluorescence signal acquisition. Compared with dual qPCR, the bacterial suspension sensitivity is 8.25 × 10⁻⁶. 2 The dual ddPCR of this invention has higher detection sensitivity, with a genomic sensitivity of 3.14 fg / μL and a bacterial culture sensitivity of up to 8.25 × 10⁻⁶ CFU / mL. 1 CFU / mL.

[0034] (4) The results of this invention are simple to determine, without the need for complicated electrophoresis operations, and the detection time is shortened to less than 3 hours, which can meet the detection needs of general laboratories. Attached Figure Description

[0035] Figure 1 Electrophoresis diagrams for verifying the specificity of self-screened targets; A: AOT11_05055 gene; B: AOT11_09030 gene; C: AOT11_17375 gene; D: AOT11_17485 gene; E: AOT11_21945 gene.

[0036] M: DL1000 DNA marker; N: Blank control; Lane 1: Vibrio vulnificus ATCC 27562; Lane 2: Vibrio vulnificus CICC 25009; Lane 3: Vibrio vulnificus isolate; Lane 4: Vibrio vulnificus isolate; Lane 5: Vibrio parahaemolyticus ATCC 33847; Lane 6: Vibrio parahaemolyticus ATCC 17802; Lane 7: Vibrio alginolyticus ATCC 17749; Lane 8: Vibrio cholerae CICC 23794; Lane 9: Salmonella typhimurium ATCC 14028; Lane 10: Escherichia coli ATCC 25922; Lane 11: Staphylococcus aureus CMCC(B)260031; Lane 12: Listeria monocytogenes ATCC 19115; Lane 13: Pseudomonas aeruginosa ATCC 9027; Lane 14: Bacillus cereus CMCC(B)63303.

[0037] Figure 2 Results of specificity and genomic sensitivity for duplex qPCR detection; A: Validation of primer and probe specificity; B: Amplification curves of duplex qPCR at different annealing temperatures (56.8℃-64.8℃), where D1~D6, 31.4~3.14×10⁻⁶. 6 fg / μL.

[0038] Figure 3 The first image shows a one-dimensional scatter plot of different annealing temperatures in duplex ddPCR. A and B are one-dimensional scatter plots of different annealing temperatures (56.8–64.8℃) for the vvhA and vv08030 genes in duplex ddPCR under high DNA concentration conditions, respectively. C and D are one-dimensional scatter plots of different annealing temperatures (56.8–64.8℃) for the vvhA and vv08030 genes in duplex ddPCR under low DNA concentration conditions, where T1–T8 are 56.8–64.8℃.

[0039] Figure 4The first image shows a one-dimensional scatter plot of different primer-probe ratios for duplex ddPCR. A and B are one-dimensional scatter plots of different primer-probe ratios (1:1.75 to 1:0.25) for the vvhA and vv08030 genes in duplex ddPCR under high DNA concentration conditions. C and D are one-dimensional scatter plots of different primer-probe ratios (1:1.75 to 1:0.25) for the vvhA and vv08030 genes in duplex ddPCR under low DNA concentration conditions, where R1 to R7 are 1:0.25 to 1:1.75.

[0040] Figure 5 The results show the genomic sensitivity of dual ddPCR detection; A and B are one-dimensional scatter plots of the vvhA and vv08030 genes in dual ddPCR, respectively, where D0-D4, 3.14-3.14×10⁻⁴. 4 fg / μL; NTC, no template control.

[0041] Figure 6 The results and standard curves for the sensitivity detection of dual ddPCR and dual qPCR bacterial cultures are shown. A and B are one-dimensional scatter plots of dual ddPCR and their corresponding standard curves, respectively. C and D are amplification curves of dual qPCR and their corresponding standard curves, respectively. B1–B7 are 8.25 × 10⁻⁶. 1 ~8.25×10 7 CFU / mL. Detailed Implementation

[0042] The present invention will be further described below with reference to the embodiments. The experimental methods in the following embodiments, unless otherwise specified, are generally carried out in accordance with known means in the art or according to the manufacturer's recommended conditions. The strains involved in the embodiments are all prior art and can be easily obtained by those skilled in the art from publicly available commercial channels.

[0043] Example 1: Discovery of Vibrio vulnificus-specific targets

[0044] Twenty-three published whole genome sequences of Vibrio vulnificus strains were downloaded from the NCBI (National Center for Biotechnology Information) public database (https: / / www.ncbi.nlm.nih.gov / ). All CDS sequences were compiled into FASTA format using the NCBI genome batch processing tool (https: / / www.ncbi.nlm.nih.gov / sites / batchentrez) to establish a local database. The standard strain ATCC 27562 was selected as the reference strain, as it has been extensively studied. Reference strain ATCC 27562 has two chromosomes and contains a total of 4326 CDS sequences.

[0045] First, the CDS sequence of the reference strain was compared with the CSD sequences of 22 other Vibrio vulnificus strains in the local database using computer programming. The parameter was set to E-value < 1e-200, and CDS sequences with a hit value (hits) of at least 22 were selected as the initial screening results. The NCBI Blasten system was then used to perform online comparisons of the eligible candidate targets. CDS sequences showing good homology within Vibrio vulnificus (E-value < 1e-200, Query Cover ≥ 98%, Identities ≥ 90%) and high specificity in non-Vibrio vulnificus species (E-value ≥ 1e-20, Query Cover < 6%) were selected as the secondary screening results. Subsequently, the NCBI BLSAT system was used to perform online comparisons of these quasi-specific targets, and the selected quasi-specific targets that met the requirements were subjected to further functional validation.

[0046] Example 2: Functional Verification of the Target

[0047] Six novel targets, AOT11_08030, AOT11_05055, AOT11_09030, AOT11_17375, AOT11_17485, and AOT11_21945, were identified through bioinformatics screening. Using the CDS sequences of targets AOT11_05055, AOT11_09030, AOT11_17375, AOT11_17485, and AOT11_21945 as templates, standard PCR primers were designed using Primer Premier 5.0 software. All primers underwent preliminary specificity verification using the Primer-BLAST online tool (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / index.cgi). The qualified primers were then sent to Nanjing Genscript Biotech Co., Ltd. The specific primer sequences are as follows:

[0048] AOT11_05055-F: 5'-GCTTTGTCTATCGTGACTTGG-3', as shown in SEQ ID NO.13;

[0049] AOT11_05055-R: 5'-CACTTTGTCCCCTTCTCCC-3', as shown in SEQ ID NO.14;

[0050] AOT11_09030-F: 5'-TACCCACGAAGTCTACGCC-3', as shown in SEQ ID NO.15;

[0051] AOT11_09030-R: 5'-TCTTGGACAAACACGCTCA-3', as shown in SEQ ID NO.16;

[0052] AOT11_17375-F: 5'-GGTTGGACTCCTGACTCTGTT-3', as shown in SEQ ID NO.17;

[0053] AOT11_17375-R: 5'-GAGCCATGTTTTCACCTTCTG-3', as shown in SEQ ID NO.18;

[0054] AOT11_17485-F: 5'-AAGCCCGATGGTTTGAATAC-3', as shown in SEQ ID NO.19;

[0055] AOT11_17485-R: 5'-TCGCTAGAGCGGCAAGATA-3', as shown in SEQ ID NO.20;

[0056] AOT11_21945-F: 5'-GCTCCTGTTCATCCTCGTG-3', as shown in SEQ ID NO.21;

[0057] AOT11_21945-R: 5'-CGGGGTTAAGTCGTGTTGT-3', as shown in SEQ ID NO.22;

[0058] Fourteen strains used for specificity verification are shown in Table 1. Eight of these Vibrio strains were inoculated into 10 mL of 3% NaCl alkaline peptone water (APW), while the remaining strains were inoculated into 10 mL of LB medium and cultured at 36℃±1℃ with shaking for 16-18 h. 1 mL of fresh culture was centrifuged at 6,000×g for 5 min, the supernatant was discarded, and the cells were resuspended in 50 μL of double-distilled water. The cells were boiled at 100℃ for 10 min, then immediately placed on ice for 10 min, and centrifuged at 14,000×g for 5 min. The supernatant was used for PCR detection or stored at -20℃ for later use.

[0059] Table 1. Target-specific validation strains and detection results

[0060]

[0061]

[0062] Note: ATCC (American Type Culture Collection); CICC (China Center of Industrial Culture Collection); CMCC (China Medical Culture Collection); "+" indicates a positive amplification result; "-" indicates a negative amplification result.

[0063] The results of the self-screening target specificity verification are as follows: Figure 1 As shown, only Vibrio vulnificus exhibited specific target bands at positions 209bp (AOT11_05055), 441bp (AOT11_09030), 366bp (AOT11_17375), 195bp (AOT11_17485), and 270bp (AOT11_21945), while no amplification bands were generated at the target positions for non-Vibrio vulnificus. These results clearly demonstrate that the self-screening target provided by this invention has high specificity, good stability, and a wide range of applications.

[0064] Example 3: qPCR based on TaqMan probe method

[0065] (1) Design primers and probes

[0066] Highly specific primers and probes were designed based on conserved sequences of the self-screened target AOT11_08030 (located on chromosome 1, accession number: CP012881.1, named vv08030) and the conventional target vvhA (located on chromosome 2, accession number: CP012882.1), respectively. The specific primer and probe sequences are as follows:

[0067] vv08030-F: 5'-CAACTACTGCGAGTGGTTTCC-3', as shown in SEQ ID NO.7;

[0068] vv08030-R: 5'-CCATGCTTCAGCGGGTCT-3', as shown in SEQ ID NO.8;

[0069] vv08030-P: 5'-HEX-ACTTGCTTGGCTCACCCGACTC-BHQ1-3', as shown in SEQ ID NO.9;

[0070] vvhA-F: 5'-CAGGTTGGCGCACAAGAAT-3', as shown in SEQ ID NO.10;

[0071] vvhA-R: 5'-CACATTGACGCGAACATCG-3', as shown in SEQ ID NO.11;

[0072] vvhA-P: 5'-FAM-CCCGCATTACACCAGTCTCGTG-BHQ1-3', as shown in SEQ ID NO.12.

[0073] This invention employs the Taqman probe method, the main principle of which is to utilize the process by which an enzyme cleaves a specific probe with fluorescent reporter groups (FAM, HEX) and a fluorescent quencher group (BHQ1) bound to the template during chain elongation, thereby generating a fluorescent signal. The intensity of the fluorescent signal reflects the template content. Compared with the dye method, the Taqman probe method has the advantage of high specificity.

[0074] (2) Specificity verification

[0075] The specificity verification strains were the same as in Example 2. Eight Vibrio strains were inoculated into 10 mL of 3% NaCl alkaline peptone water (APW), and the remaining strains were inoculated into 10 mL of LB medium. The cultures were incubated at 36℃±1℃ with shaking for 16-18 h. 1 mL of fresh culture was centrifuged at 6,000×g for 5 min, the supernatant was discarded, and the cells were resuspended in 50 μL of double-distilled water. The cells were boiled at 100℃ for 10 min, then immediately placed on ice for 10 min, and centrifuged at 14,000×g for 5 min. The supernatant was used for PCR detection or stored at -20℃ for later use.

[0076] Dual qPCR reaction system (25 μL): 12.5 μL Premix Ex Taq (Probe qPCR), 1.0 μL each of upstream and downstream primers (10 μmol / L) for vvhA and vv08030 genes, 0.5 μL probe (10 μmol / L), 5 μL DNA template, and finally water to make up to 25 μL.

[0077] Dual qPCR amplification conditions: 95℃ for 30s; 95℃ for 5s, 60℃ for 30s (collecting FAM and HEX fluorescence), 40 cycles.

[0078] Specificity verification results are as follows Figure 2 As shown in Figure A, only Vibrio vulnificus showed the expected amplification curve, while no amplification curves were generated for any non-Vibrio vulnificus.

[0079] (3) Optimization of annealing temperature for dual qPCR

[0080] 3.14×10 7 DNA template was serially diluted 10-fold to obtain 10 fg / μL DNA template. 6 10 5 10 4 10 3 10 2 DNA solutions of 10 fg / μL were labeled D6–D1, and optimization was performed at different annealing temperatures (56.8℃–64.8℃). Figure 2 B shows that within the annealing temperature range of 56.8℃ to 64.8℃, the amplification efficiency of both vvhA and vv08030 genes is 90% to 100%, exhibiting good linearity (R). 2 The result showed a genomic sensitivity of 31.4 fg / μL (>0.99). These results demonstrate that the method provided by this invention has the advantages of good specificity, repeatability, stability, and sensitivity.

[0081] Example 4: Optimization of Dual ddPCR Amplification Conditions and Reaction System

[0082] Based on the recommended system and procedures in the instruction manual, we optimized the annealing temperature and screened the primer-probe ratio.

[0083] Dual ddPCR reaction system (20 μL): 10 μL ddPCR Supermix for Probes (No dUTP), 1.0 μL each of upstream and downstream primers (10 μmol / L) for vvhA and vv08030 genes, 0.5 μL probe (10 μmol / L), 4 μL DNA template, and finally water to make up to 20 μL.

[0084] Amplification conditions: 95℃ for 10 min; 94℃ for 30 s; 60℃ for 1 min, 40 cycles; 98℃ for 10 min; store at 4℃ (the temperature rise and fall rate for all temperatures in the program is 2℃ / s). After amplification, read the values ​​using a QX200 Droplet Reader.

[0085] (1) Annealing temperature optimization: Annealing temperature is an important factor affecting PCR specificity. ddPCR amplification reactions were performed at different annealing temperatures (56.8℃, 57.3℃, 58.4℃, 60.0℃, 61.9℃, 63.5℃, 64.4℃, 64.8℃). The optimal annealing temperature was selected based on the good separation between positive and negative droplets and the high fluorescence signal value.

[0086] Depend on Figure 3 It can be seen that, regardless of the template DNA concentration, the positive and negative droplets of the vvhA and vv08030 genes can be clearly separated into two clusters at different annealing temperatures (56.8℃~64.8℃). However, at higher template DNA concentrations and lower annealing temperatures (56.8℃, 57.3℃), the number of diffuse droplets between the positive and negative droplets of the vvhA gene is greater. Considering both DNA concentration and fluorescence signal value, 60℃ was selected as the optimal annealing temperature.

[0087] (2) Primer-probe ratio optimization: While ensuring the differentiation effect between positive and negative droplets, seven different primer-probe ratios (1:1.75, 1:1.5, 1:1.25, 1:1, 1:0.75, 1:0.5, 1:0.25) were set to determine the optimal primer-probe ratio and reduce detection costs.

[0088] Depend on Figure 4 It was observed that, regardless of the template DNA concentration, the fluorescence signal of positive droplets increased with increasing probe concentration, but the fluorescence signal of negative droplets also increased synchronously, indicating an increase in fluorescence background. This suggests that simply increasing the probe content in the system to improve the signal response has no practical effect on improving the resolution of ddPCR. Considering both resolution and detection cost, a primer-probe ratio of 1:0.5 was selected as the optimal ratio.

[0089] Example 5: Sensitivity Evaluation of the Detection System

[0090] This invention detects genomic DNA templates and fresh bacterial culture templates to determine their sensitivity.

[0091] (1) Genomic DNA template: Using the genomic DNA of Vibrio vulnificus standard strain (ATCC 27562) as a template, 10-fold serial dilutions were performed to obtain 10 4 10 310 2 10 1 10 0 DNA solutions of fg / μL were amplified using the established doublet ddPCR method.

[0092] The detection sensitivity of dual ddPCR was found to be 3.14 fg / μL when genomic DNA was used as a template (see [link to relevant documentation]). Figure 5 ).

[0093] (2) Bacterial culture template: Using the culture medium of Vibrio vulnificus standard strain (ATCC 27562) as a template, 10-fold serial dilutions were performed to obtain 10 0 CFU / mL ~10 7 CFU / mL bacterial culture was amplified using the established dual ddPCR and dual qPCR methods.

[0094] When using bacterial culture as a template, bacterial cultures with different dilution gradients were simultaneously plated. The results showed that the original bacterial culture concentration was 8.25 × 10⁻⁶. 8 CFU / mL.

[0095] Depend on Figure 6 It can be seen that when using bacterial culture as a template, the sensitivity of dual ddPCR detection is 8.25 × 10⁻⁶. 1 CFU / mL, the corresponding copy numbers of the vvhA gene and the vv08030 gene were 3.67 copies / 20 μL and 3.33 copies / 20 μL, respectively. The standard curve equation is log 10 copies / mL = 0.9928 log 10 CFU / mL +0.0602 (vvhA gene, R) 2 =0.9993), log 10 copies / mL = 0.9952 log 10 CFU / mL +0.0570 (vv08030 gene, R) 2 =0.9989);

[0096] The sensitivity of the dual qPCR detection is 8.25 × 10⁻⁶. 2 CFU / mL, the standard curve equation is Cq = -3.202log 10 CFU / mL +44.43 (vvhA gene, R) 2 =0.9986), Cq = -3.189log 10 CFU / mL +44.44 (vv08030 gene, R) 2 =0.9995).

[0097] Therefore, the dual ddPCR method provided by this invention has higher detection sensitivity than the dual qPCR method.

[0098] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. The application of primer sets in the preparation of detection kits or reagents for Vibrio vulnificus, characterized in that, The primer set includes primer set A, which specifically targets the gene fragment AOT11_08030, and primer set B, which specifically targets the vvhA gene. The primer set A includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8, and a probe with a nucleotide sequence as shown in SEQ ID NO.9; The primer set B includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.10, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.11, and a probe with a nucleotide sequence as shown in SEQ ID NO.

12.

2. The application according to claim 1, characterized in that, The probe is modified with a fluorescent reporter group at its 5' end and a quencher group at its 3' end.

3. The application according to claim 2, characterized in that, The reporter group is selected from, but not limited to, FAM, HEX, JOE, CY3, and CY5, and the quencher group is selected from, but not limited to, DABCYL, TAMRA, and BHQ.

4. The application according to claim 2 or 3, characterized in that, The 5' end of the probe in primer set A is modified with the fluorescent reporter group HEX.

5. The application according to claim 2 or 3, characterized in that, The 5' end of the probe of primer set B is modified with a fluorescent reporter group FAM.

6. A kit for detecting Vibrio vulnificus, characterized in that, It contains primer set A, which specifically targets the gene fragment AOT11_08030, and primer set B, which specifically targets the vvhA gene. The primer set A includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.7, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.8, and a probe with a nucleotide sequence as shown in SEQ ID NO.9; The primer set B includes an upstream primer with a nucleotide sequence as shown in SEQ ID NO.10, a downstream primer with a nucleotide sequence as shown in SEQ ID NO.11, and a probe with a nucleotide sequence as shown in SEQ ID NO.12.

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