A primer probe set, method and application for simultaneous detection of Vibrio parahaemolyticus and drug-resistant genes

Abstract

The present invention discloses a primer-probe set, method, and application for the simultaneous detection of Vibrio parahaemolyticus and drug-resistant genes, belonging to the technical field of food safety detection. Through multiple rounds of screening, the present invention obtained a primer-probe combination suitable for rapid, parallel detection of dual targets. Combining the advantages of RPA isothermal amplification and LFD stability and convenience, a dual RPA-LFD detection method for Vibrio parahaemolyticus was established, with a detection limit of 2.04×10 2 The present invention has simple operation steps, short detection cycle, and low equipment dependence, filling the gap in the current method for simultaneous detection of Vibrio parahaemolyticus and drug-resistant genes, and can meet the needs of rapid on-site detection and intuitive visualization of food safety.

Description

Technical Field

[0001] The present invention relates to a primer probe set, method and application for simultaneously detecting Vibrio parahaemolyticus and drug-resistant genes, belonging to the technical field of food safety detection. Background Art

[0002] Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) is a Gram-negative halophilic bacterium widely distributed in marine environments and aquatic products. Currently, detection of Vibrio parahaemolyticus primarily relies on the test method outlined in the current national standard GB4789.7-2013. The general steps include pre-enrichment, isolation on selective culture media, pure culture, biochemical reactions, and serotyping, with the entire process typically taking 5–7 days. For decades, the use of antimicrobial agents in aquaculture and clinical treatment has been a long-standing practice, leading to the development of drug resistance. Regarding the detection and assessment of drug resistance, the recommended methods by the US CLSI and the EUCAST are broth microdilution and disc diffusion, but these methods typically require approximately 18 hours of incubation. Drug resistance is considered a stable biological phenotype of pathogenic microorganisms and is generally directly associated with the acquisition of resistance genes. Tetracycline is widely used as a veterinary drug in livestock and aquaculture for disease prevention, treatment, and growth promotion, resulting in high residual concentrations in food and the environment. Previous research by our team has found that subinhibitory concentrations of tetracycline can promote the development of resistance genes. tetA Therefore, rapid identification of pathogenic microorganisms and their resistance results can provide a scientific basis for formulating timely and effective prevention and control strategies, which is of great significance for further safeguarding public health and food safety.

[0003] Isothermal amplification (RPA) allows for rapid DNA amplification at a constant temperature, eliminating the need for time-consuming thermal cycling steps. Compared to loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA) offers the advantages of fewer primers, lower reaction temperatures, and shorter detection times. To further simplify the protocol and improve the portability and stability of the assay, lateral flow dipsticks (LFDs) offer significant advantages for interpreting RPA results. However, whether and how dual RPA-LFD can be used for the simultaneous detection of V. parahaemolyticus and drug-resistance genes remains unknown. Therefore, the present invention proposes a primer-probe set, method, and application for the simultaneous detection of V. parahaemolyticus and drug-resistance genes based on dual RPA-LFD. Summary of the Invention

[0004] In response to the deficiencies in the above-mentioned prior art, the present invention provides a dual RPA-LFD primer probe group, method and application for the simultaneous detection of Vibrio parahaemolyticus and drug-resistant genes. First, a single RPA-LFD primer pair test is conducted, and then a dual RPA-LFD multiple primer pair combination test is conducted to screen and obtain the optimal primer probe combination. Then, the reaction system and reaction procedure are optimized, and finally a new dual RPA-LFD detection technology for the simultaneous detection of Vibrio parahaemolyticus and drug-resistant genes is successfully constructed. The purpose is to fill the gap in the current simultaneous detection method of Vibrio parahaemolyticus and drug-resistant genes, and it has good application prospects in on-site rapid detection.

[0005] The first technical solution provided by the present invention is a composition, which comprises two sets of primer probes; the first set of primer probes is used to specifically detect Vibrio parahaemolyticus, including a first upstream primer, a first downstream primer and a first probe, the first upstream primer sequence is shown as SEQ ID NO: 1, the first downstream primer sequence is shown as SEQ ID NO: 3, and the first probe sequence is shown as SEQ ID NO: 5; the second set of primer probes is used to detect drug-resistant genes, including a second upstream primer, a second downstream primer and a second probe, the second upstream primer sequence is shown as SEQ ID NO: 6, the second downstream primer sequence is shown as SEQ ID NO: 9, and the second probe sequence is shown as SEQ ID NO: 13.

[0006] In certain embodiments, the 5' end of the first upstream primer is modified with biotin, the 5' end of the first probe is modified with 6-hydroxyfluorescein, the 3' end of the first probe is modified with Spacer C3, and the first probe is modified with dSpacer (THF) between nucleotides 29 and 30.

[0007] In certain embodiments, the 5' end of the second downstream primer is modified with biotin, the 5' end of the second probe is modified with digoxigenin, the 3' end of the second probe is modified with Spacer C3, and the second probe is modified between nucleotides 33 and 34 with dSpacer (THF).

[0008] The second technical solution provided by the present invention is a kit for simultaneously detecting Vibrio parahaemolyticus and drug-resistant genes, wherein the kit comprises the composition described in the first technical solution.

[0009] In certain embodiments, the kit further comprises an RPA amplification reagent and a lateral flow chromatography test strip.

[0010] In certain embodiments, the RPA amplification reagents include AD buffer, B buffer, and ultrapure water.

[0011] In certain embodiments, the lateral flow chromatography test strip is provided with a T1 detection line and a T2 detection line, which are used to capture 6-hydroxyfluorescein and digoxin, respectively.

[0012] In certain embodiments, the kit further comprises a positive control substance of Vibrio parahaemolyticus carrying a drug-resistant gene.

[0013] The third technical solution provided by the present invention is a method for simultaneously detecting Vibrio parahaemolyticus and drug-resistant genes for non-disease diagnosis purposes, wherein the method comprises detecting Vibrio parahaemolyticus using the kit described in the second technical solution.

[0014] In certain embodiments, the method comprises the steps of:

[0015] (1) Extract genomic DNA from the sample to be tested or directly dilute the sample to be tested;

[0016] (2) constructing an RPA reaction system using the composition described in the first technical solution, performing isothermal amplification, and obtaining an amplified product;

[0017] (3) Drop the amplified product obtained in step (2) onto the sample loading hole of the lateral flow chromatography test strip to obtain the test result.

[0018] In certain embodiments, in step (2), the total volume of the RPA reaction system is 50 μL, AD buffer 29.4 μL, 10 μM first upstream and downstream primers are added 2 μL each, 10 μM first probe is added 0.6 μL, 10 μM second upstream and downstream primers are added 1 μL each, 10 μM second probe is added 0.3 μL, sample 5 μL, B buffer 2.5 μL, and ddH2O is added to make up to 50 μL; the amplification program is 40°C, 25 min.

[0019] In certain embodiments, in step (3), 7 μL of the amplified product obtained in step (2) is added to 98 μL of sterile ddH2O, mixed, and then 80 μL is dropped onto the sample well of the lateral flow chromatography test strip, and the test result is interpreted within 5-10 minutes.

[0020] The fourth technical solution provided by the present invention is the use of the composition described in the first technical solution or the second technical solution in detecting Vibrio parahaemolyticus and / or Vibrio parahaemolyticus containing drug-resistant genes.

[0021] Compared with the prior art, the technical effects of the present invention are as follows:

[0022] (1) The present invention screens the primers and probes of specific genes and drug-resistant genes step by step through preliminary experiments. vps2310Among the three primer-probe combinations of the gene, combination 3 that produced false positives was excluded; tetA Of the six primer-probe combinations for the gene, combinations 2, 5, and 6, which produced false positives, were excluded. Based on the above single-gene results, six pairwise combination schemes (2×3) could be generated, but ultimately only one primer combination 2-3 for dual-target detection was obtained, which ensured accurate sample detection in the dual RPA-LFD reaction system.

[0023] (2) The present invention combines the advantages of RPA isothermal amplification and LFD convenience and rapidity to establish a dual RPA-LFD rapid detection method for Vibrio parahaemolyticus, which has the advantages of low equipment requirements and intuitive visualization. Among the 40 experimental strains, 7 isolates of Vibrio parahaemolyticus carrying drug-resistant genes showed double bands, and 22 isolates of Vibrio parahaemolyticus not carrying drug-resistant genes only showed double bands at the T1 detection line ( vps2310 A single band appeared at the gene, and all 11 negative strains of non-V. parahaemolyticus showed only the quality control line band, indicating good specificity.

[0024] (3) This invention fills the gap in the simultaneous detection method of Vibrio parahaemolyticus and drug-resistant genes. The detection can be completed within 30-35 minutes, and the detection limit for pure culture of Vibrio parahaemolyticus is 2.04×10 2 CFU / mL, which is very suitable for rapid on-site testing and helps to identify potential high-risk drug-resistant bacteria at an early stage. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Optimize the reaction temperature for double RPA; 1 to 4 are 37 ℃, 40 ℃, 43 ℃, and 45 ℃, respectively.

[0026] Figure 2 Optimized for dual RPA primer ratio; A: vps2310 and tetA The primer ratio of the gene is 0.5:1; B: vps2310 and tetA The primer ratio of the gene is 1:1; C: vps2310 and tetA The primer ratio of the gene is 2:1; D: vps2310 and tetA The ratio of primers for the genes was 4:1; 1 to 3 were 10 2 , 10 1 , 10 0 CFU / mL.

[0027] Figure 3 For the optimization of double RPA reaction time; A: 15 min; B: 20 min; C: 25 min; D: 30 min; 1 to 3 are 10 2, 10 1 , 10 0 CFU / mL.

[0028] Figure 4 The results of the double RPA-LFD method are specific. 1 to 7 are the Vibrio parahaemolyticus isolates NJIFDCVp52, NJIFDCVp19, NJIFDCVp20, NJIFDCVp21, NJIFDCVp40, NJIFDCVp147, and NJIFDCVp161 carrying drug-resistant genes; 8 to 11 are the standard strains ATCC 17802, ATCC 33847, CICC 21619, and CICC 10552; 12 to 29 are the Vibrio parahaemolyticus isolates NJIFDCVp1 to NJIFDCVp18 not carrying drug-resistant genes; 30 to 40 are Vibrio alginolyticus ATCC 17749, Vibrio cholerae CICC 23794, Vibrio vulnificus ATCC 27562, Staphylococcus aureus ATCC 25923, and Salmonella typhimurium ATCC 14028, Shigella flexneri CMCC (B) 51572, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19115, Cronobacter sakazakii ATCC 29544, Bacillus cereus CMCC(B) 63303, and Pseudomonas aeruginosa ATCC 9027.

[0029] Figure 5 The sensitivity evaluation results of the dual RPA-LFD method are shown in Figure 2. Among them, 1 to 8 are 2.04×10 7 , 2.04×10 6 , 2.04×10 5 , 2.04×10 4 , 2.04×10 3 , 2.04×10 2 , 2.04×10 1 , 2.04×10 0 CFU / mL.

[0030] Figure 6 Results of single-plex RPA-LFD primer screening; A: vps2310 Gene primer combination number 1; B: vps2310 Gene primer combination number 2; C: vps2310 Gene primer combination number 3; D: tetA Gene primer combination number 1; E: tetA Gene primer combination number 2; F: tetA Gene primer combination number 3; G: tetA Gene primer combination number 4; H: tetA Gene primer combination number 5; I: tetA Gene primer combination number 6.

[0031] Figure 7 Results of dual RPA-LFD primer screening; A: combination number 1-1; B: combination number 1-3; C: combination number 1-4; D: combination number 2-1; E: combination number 2-3; F: combination number 2-4. DETAILED DESCRIPTION

[0032] The following describes preferred embodiments of the present invention. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.

[0033] The raw materials used in the embodiment are:

[0034] The main reagents and culture media used in the following examples: DNA isothermal amplification kit (colloidal gold test strip type) and dual nucleic acid detection test strips (latex chromatography method) were purchased from Anpu Future Biotechnology Co., Ltd.; 3% sodium chloride alkaline peptone water (APW) and LB broth were purchased from Beijing Luqiao Technology Co., Ltd.

[0035] The main instruments and equipment used in the following examples are: CFX96 real-time fluorescence quantitative PCR instrument (Bio-Rad, USA); KB240 low-temperature incubator (Binder, Germany); TS-200B constant temperature shaker (Shanghai Tiancheng Laboratory Instrument Manufacturing Co., Ltd.); 1-14 tabletop centrifuge (Sigma, Germany); Bagmixer 400 slap-type homogenizer (Interscience, France); 1300 series A2 biological safety cabinet (Thermo Fisher Scientific, USA).

[0036] Example 1: Establishment of RPA detection method

[0037] 1.1 RPA primer and probe design

[0038] Selection of Vibrio parahaemolyticus-specific target genes ( vps2310 ) and tetracycline resistance genes ( tetA ) as the detection target, and RPA primers and probes were designed using Primer Premier 5.0 software according to RPA primer design principles. All primers and probes were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The specific primer sequences are as follows:

[0039] vps2310 -F (first upstream primer): 5'-Biotin-ACATTTCGAGTAGGTAGCTGATGCCAAGC-3' (SEQ ID NO: 1);

[0040] vps2310 -R (first downstream primer): 5′-TTTTGGCTCTATGACTTCCGTTTATCCCT-3′ (SEQ ID NO: 3);

[0041] vps2310 -P: (first probe) 5'-FAM-CCATTCTTCTCGTGATTAACTGGTATTGG / dSpacer(THF) / TGAAGGAAGGGCAAA-3'-Spacer C3 (SEQ ID NO: 5);

[0042] tetA -F (second upstream primer): 5′-TTTCGGCGAGGATCGCTTTCACTGGGACG-3′ (SEQ ID NO: 6);

[0043] tetA -R (second downstream primer): 5'-Biotin-CAGCGCCTGCAATCCATGCCCACCCGTTCC-3' (SEQ ID NO: 9);

[0044] tetA -P (secondary probe): 5′-Digoxigenin-CGAAAGGCGGGCACTCATGCTCGGAATGATTGC / dSpacer(THF) / GACGGCACAGGCTACA-3′-Spacer C3 (SEQ ID NO: 13).

[0045] Add the above primers and probes to the following system for reaction: 29.4 μL AD buffer, vps2310 Gene primers vps2310 -F (10 μM), vps2310 -R (10 μM) were added 1 μL, vps2310 Add 0.3 μL of gene probe (10 μM), tetA Gene primers tetA -F (10 μM), tetA -R (10 μM) were added 1 μL, tetA Add 0.3 μL of gene probe (10 μM), 5 μL of nucleic acid to be tested, and 2.5 μL of buffer B. Add ddH2O to make up to 50 μL. Reaction conditions: 40°C, 25 min.

[0046] 1.2 Reaction conditions and system optimization

[0047] Reaction temperature optimization: The experimental setting temperature was 37 ℃, 40 ℃, 43 ℃, and 45 ℃. Figure 1 It can be seen that when the reaction temperature is 37 ℃ and 45 ℃, vps2310 The gene shows a band at the T1 detection line. tetA No bands appeared at the T2 detection line for the gene. Bands appeared at both the T1 and T2 detection lines at reaction temperatures of 40°C and 43°C. At 40°C, there was no significant difference in the brightness of the two detection lines, so 40°C was selected as the optimal reaction temperature for dual RPA.

[0048] Dual RPA primer ratio optimization: Consider the differences in amplification efficiency of different primers and set vps2310 and tetA The primer ratios of the genes were 0.5:1, 1:1, 2:1, and 4:1. At the same time, considering the sensitivity of the method, the primer ratios of the genes were 0.5:1, 1:1, 2:1, and 4:1. 2 , 10 1 , 10 0 CFU / mL) range to optimize the primer ratio. Figure 2 As shown, when the primer ratio is 0.5:1, 2 CFU / mL bacterial solution concentration, a weak double band appears ( Figure 2 A in ); when the primer ratio is 1:1, the T2 detection line ( tetA gene) is brighter, and at 10 1 CFU / mL bacterial solution concentration showed a weak band ( Figure 2 B in the figure); when the primer ratio is 2:1, 2 CFU / mL bacterial solution concentration, visible double bands appear ( Figure 2 C in Figure 1); When the primer ratio was further increased to 4:1, only the T1 detection line was at 10 2 A single band appeared at a concentration of CFU / mL. Considering the brightness of the band and the detection sensitivity, 2:1 was selected as vps2310 and tetA Optimal primer ratio for each gene.

[0049] Reaction time optimization: Set the reaction time to 15 min, 20 min, 25 min, and 30 min. Figure 3 As shown, with the increase of reaction time, the T1 detection line ( vps2310 The brightness of the band of gene) gradually increased, and the T2 detection line ( tetA The band brightness of the gene (gene) showed a trend of increasing first and then decreasing. Taking into account band brightness and detection sensitivity, 25 minutes was selected as the optimal reaction time for dual RPA.

[0050] 1.3 Determining the optimal detection conditions for dual RPA-LFD

[0051] The reaction system is: AD buffer 29.4 μL, vps2310 Gene primers vps2310 -F (10 μM), vps2310 -R (10 μM) were added 2 μL, vps2310 Add 0.6 μL of gene probe (10 μM), tetA Gene primers tetA -F (10 μM), tetA -R (10 μM) were added 1 μL, tetA Add 0.3 μL of the gene probe (10 μM), 5 μL of the nucleic acid to be tested, and 2.5 μL of buffer B. Add ddH2O to a volume of 50 μL. The reaction conditions are: 40°C, 25 min. After the reaction is complete, add 7 μL of the amplified product to 98 μL of sterile ddH2O. Mix thoroughly, then dispense 80 μL onto the sample well of a lateral flow chromatography test strip. Read the test results within 5-10 min.

[0052] Example 2: Dual RPA-LFD specificity verification

[0053] The dual RPA-LFD method established in Example 1 was used as the template for detection using the genomic DNA of 29 positive strains of Vibrio parahaemolyticus and 11 negative strains of non-Vibrio parahaemolyticus (Table 1) preserved in our laboratory.

[0054] Table 1 Strains used for specificity evaluation and test results

[0055]

[0056] Note: ATCC (American Type Culture Collection), American Type Culture Collection; CICC (China Center of Industrial Culture Collection), China Center for Industrial Culture Collection; CMCC (China Medical Culture Collection); NJIFDCVp1-21, NJIFDCVp40, NJIFDCVp52, NJIFDCVp147, and NJIFDCVp161 represent 25 isolates of Vibrio parahaemolyticus.

[0057] Method-specific test results such as Figure 4 As shown, 7 isolates of Vibrio parahaemolyticus carrying drug-resistant genes showed double bands, while 4 standard strains of Vibrio parahaemolyticus and 18 isolates of Vibrio parahaemolyticus not carrying drug-resistant genes showed double bands only at the T1 detection line ( vps2310A single band appeared at the site of the tetracycline resistance gene, while all 11 non-negative strains of Vibrio parahaemolyticus only showed the quality control line band, indicating that the established dual RPA-LFD method can simultaneously detect Vibrio parahaemolyticus and tetracycline resistance genes, and has no cross-reaction with other negative bacteria, with good specificity.

[0058] Example 3: Dual RPA-LFD sensitivity test

[0059] Take the bacterial solution cultured to the logarithmic growth phase and dilute it tenfold with sterile saline to 10 -8 Each gradient dilution was boiled to extract DNA, and used as a template for double RPA-LFD detection to determine the minimum detection limit of the method. -5 , 10 -6 and 10 -7 Three dilutions were counted and the experiment was set up in triplicate.

[0060] In the sensitivity evaluation of the dual RPA-LFD method, the concentration of Vibrio parahaemolyticus was 2.04×10 8 CFU / mL, the concentration obtained by serial dilution was 2.04×10 7 ~2.04 CFU / mL of bacterial solution. Figure 5 As shown, as the bacterial concentration decreases, the T1 detection line ( vps2310 The brightness of the band of gene) showed a trend of first increasing and then decreasing. 4 The brightness is the highest when the bacterial solution concentration is CFU / mL; as the bacterial solution concentration decreases, the T2 detection line ( tetA The brightness of the band of gene) gradually weakened. When the concentration of bacterial solution decreased to 10 1 CFU / mL, no visible bands appeared on the T1 and T2 detection lines. Therefore, the detection limit of the dual RPA-LFD method was 2.04×10 2 CFU / mL.

[0061] Example 4: Detection of artificially contaminated samples

[0062] Prepare salmon that has tested negative for Vibrio parahaemolyticus according to GB 4789.7-2013 "National Food Safety Standard for Microbiological Examination of Foods for Vibrio parahaemolyticus". Weigh 25 g of the sample and add 10 2 ~10 8Prepare artificial contamination samples by adding 2.5 mL of each suspension containing 100 CFU / mL of Vibrio parahaemolyticus. Add 225 mL of 3% sodium chloride alkaline peptone water to the artificially contaminated samples and homogenize to a 1:10 sample solution (the bacterial solution dilution ratio is 1:100). Incubate at 36°C ± 1°C for 0, 2, and 4 hours. Transfer the homogenate to a 1.5 mL centrifuge tube for dual RPA-LFD analysis.

[0063] As shown in Table 2, when no culture enrichment was performed, the minimum initial inoculum size that could be detected by the dual RPA-LFD method was 2.60×10 3 CFU / mL of target bacteria, it can be seen that the detection limit of the actual sample is 1 order of magnitude higher than that of the pure culture, which may be due to the interference of the complex matrix of the salmon sample; when the culture was enriched for 2 h and 4 h, the initial inoculum size was 2.60×10 2 CFU / mL of salmon samples can be detected. Therefore, in food sample testing, the appropriate pre-enrichment step can be selected according to the actual situation.

[0064] Table 2 Test results of artificially contaminated samples

[0065]

[0066] Note: “+” indicates detection; “-” indicates non-detection.

[0067] Comparative Example 1: Single-plex RPA-LFD primer screening

[0068] against vps2310 The 5' end of the probe was labeled with 6-hydroxyfluorescein (6-FAM), the middle position was labeled with dSpacer (tetrahydrofuran, THF), and the 3' end was labeled with a blocking group (Spacer C3); the 5' end of the upstream primer was labeled with biotin; including the set of primers in Example 1, a total of 3 sets of downstream primers were designed. tetA The 5' end of the probe was labeled with digoxigenin, the middle position was labeled with dSpacer (tetrahydrofuran, THF), and the 3' end was labeled with a blocking group (Spacer C3); the 5' end of the downstream primer was labeled with Biotin; including the set of primers in Example 1, a total of 6 sets of downstream primers were designed. Among them, the upstream primer vps2310 -F and probe vps2310 -P and downstream primers vps2310 -R1, vps2310 -R2, vps2310 -R3 are used in combination, and the numbers are vps2310 -Combination 1, vps2310 - Combination 2 (the first upstream and downstream primers in Example 1), vps2310 - Combination 3; upstream primer tetA -F and probe tetA -P and downstream primers tetA -R1, tetA -R2, tetA -R3, tetA -R4, tetA -R5, tetA -R6 are used in combination, and the numbers are tetA -Combination 1, tetA -Combination 2, tetA - Combination 3 (the second upstream and downstream primers in Example 1), tetA -Combination 4, tetA -Combination 5, tetA - Combination 6. All primers and probes were synthesized by Nanjing GenScript Biotechnology Co., Ltd., and the specific sequences are shown in Table 3.

[0069] Table 3 Primers and probes used for RPA-LFD detection

[0070]

[0071] For a single vps2310 genes, compared with primer combination 1 ( Figure 6 A) and combination 2 ( Figure 6 B), primer combination 3 ( Figure 6 C) The positive control detection line is weak, and the no-template control of primer combination 3 has a band (false positive result), so combinations 1 and 2 are selected as vps2310 Primer pairs for single-plex RPA-LFD detection of genes. tetA The detection line brightness of the positive control of the six primer combinations was similar, but the primer combination 2 ( Figure 6 E), combination 5 ( Figure 6 H) and combination 6 ( Figure 6 The no-template control of I) showed a band (false positive result), so combination 1 ( Figure 6 D), combination 3 ( Figure 6 F) and combination 4 ( Figure 6 G) as tetA Primer pairs for singleplex RPA-LFD detection of genes.

[0072] Comparative Example 2: Effects of different primer-probe combinations on dual LFD detection results

[0073] In Comparative Example 1, vps2310 and tetA The single RPA-LFD primer pairs for the gene have been screened, but the results of their combination are not yet known. vps2310Gene combination 1, combination 2 and tetA Gene combinations 1, 3, and 4 were then combined in pairs. The specific scheme is shown in Table 4.

[0074] Table 4 Primer-probe combination scheme for dual RPA-LFD detection

[0075]

[0076] The results of dual RPA-LFD detection of two target gene primer probe combinations are as follows Figure 7 As shown. The detection lines of the 6 two-combination positive controls all showed double bands, but the combination 1-1 ( Figure 7 A), combination 1-3 ( Figure 7 B), combination 1-4 ( Figure 7 C), combination 2-1 ( Figure 7 D) and combinations 2-4 ( Figure 7 F) The no-template control showed obvious bands (false positive result), so combinations 2-3 were selected ( Figure 7 E), i.e., the first upstream and downstream primers and the second upstream and downstream primers in Example 1, as the optimal primer combination for dual RPA-LFD detection.

[0077] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for simultaneous detection of Vibrio parahaemolyticus and drug resistance genes tetA The composition is characterized in that Contains two sets of primer probes; The first set of primer probes includes a first upstream primer, a first downstream primer, and a first probe, wherein the first upstream primer sequence is shown in SEQ ID NO: 1, the first downstream primer sequence is shown in SEQ ID NO: 3, and the first probe sequence is shown in SEQ ID NO: 5; The second set of primer probes includes a second upstream primer, a second downstream primer and a second probe. The second upstream primer sequence is shown in SEQ ID NO:6, the second downstream primer sequence is shown in SEQ ID NO:9, and the second probe sequence is shown in SEQ ID NO:

13.

2. The composition according to claim 1, characterized in that The 5' end of the first upstream primer is modified with biotin, the 5' end of the first probe is modified with 6-hydroxyfluorescein, the 3' end of the first probe is modified with Spacer C3, and the first probe is modified between nucleotides 29 and 30 with dSpacer; The 5' end of the second downstream primer is modified with biotin, the 5' end of the second probe is modified with digoxigenin, the 3' end of the second probe is modified with Spacer C3, and the spacer between the 33rd and 34th nucleotides of the second probe is modified with dSpacer.

3. A method for simultaneous detection of Vibrio parahaemolyticus and drug resistance genes tetA The kit is characterized in that The kit comprises the composition according to any one of claims 1 to 2.

4. The kit according to claim 3, wherein The kit also includes an RPA amplification reagent and a lateral flow chromatography test strip.

5. The kit according to claim 3 or 4, characterized in that The kit also contains a drug-resistant gene tetA parahaemolyticus positive control.

6. A method for simultaneous detection of Vibrio parahaemolyticus and drug resistance genes for non-disease diagnosis purposes tetA The method is characterized in that The method comprises the step of detecting Vibrio parahaemolyticus using the kit according to any one of claims 3 to 5.

7. The method according to claim 6, characterized in that The method comprises the following steps: (1) Extract genomic DNA from the sample to be tested or directly dilute the sample to be tested; (2) constructing an RPA reaction system using the composition, performing isothermal amplification, and obtaining an amplified product; (3) Drop the amplified product obtained in step (2) onto the sample loading hole of the lateral flow chromatography test strip to obtain the test result.

8. Use of the composition according to claim 1 or 2 or the kit according to any one of claims 3 to 5 in detecting Vibrio parahaemolyticus, characterized in that: The application is not for disease diagnosis purposes.

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