A method and application of detecting Vibrio parahaemolyticus based on prokaryotic Argonaute protein

Abstract

The present invention discloses a method and application for detecting Vibrio parahaemolyticus based on prokaryotic Argonaute protein, belonging to the technical field of food safety detection. The present invention screens gDNAs and fluorescent probes with high cutting efficiency, and combines LAMP high-efficiency amplification and Pf Taking advantage of the Ago targeted cleavage, a LAMP-based assay was established for Vibrio parahaemolyticus Pf The Ago rapid detection method effectively compensates for the high false positives of the simple LAMP technology and further improves the specificity and sensitivity of the detection. This method is easy to operate, requires low equipment, and can complete the detection within 45 minutes. The detection limit of the present invention for pure culture can reach 5.47 CFU / mL, and the detection limit for artificially contaminated samples is 7.84×10

Description

Technical Field

[0001] The invention belongs to the technical field of food safety detection, and relates to a method and application of detecting Vibrio parahaemolyticus based on prokaryotic Argonaute protein. Background Art

[0002] Vibrio parahaemolyticus ( Vibrio parahaemolyticus, Vp ) is a Gram-negative halophilic bacterium. Eating raw or undercooked aquatic products is very likely to cause infection with Vibrio parahaemolyticus. The typical clinical symptoms of Vibrio parahaemolyticus are gastroenteritis, diarrhea, etc., and in severe cases, it can lead to sepsis. At present, the main methods for detecting Vibrio parahaemolyticus generally include pre-enrichment, selective culture medium separation, pure culture, biochemical reaction and serotyping. The whole process generally takes 5 to 7 days.

[0003] The development of nucleic acid molecular detection technology has greatly shortened the detection cycle of foodborne pathogens, but PCR detection requires expensive thermal cycling equipment, which limits the application of this technology to a certain extent. Isothermal amplification technology can complete the rapid and effective amplification of DNA at a constant temperature without the need for time-consuming thermal cycling steps. Compared with other nucleic acid amplification technologies, loop-mediated isothermal amplification (LAMP) has the advantages of being fast, efficient, low-cost and does not require dedicated equipment. At present, the detection methods of LAMP products mainly include electrophoresis, turbidity, and dye methods, which have problems such as long time consumption, false positives, and low specificity. In recent years, a variety of nucleic acid detection methods have been developed using programmable nuclease technology, namely diagnostic technology based on the CRISPR / Cas system. However, there are still many problems with CRISPR / Cas technology, such as the high cost and instability of guide RNA, the inherent off-target nature of Cas effector proteins, and the strict reliance on the targeting mechanism of the protospacer sequence adjacent motif, which largely limits its scope of application. Therefore, it is urgent to develop a Vibrio parahaemolyticus detection method that can meet the detection needs of both on-site and laboratory and can further improve the detection specificity and sensitivity. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides a method and application for detecting Vibrio parahaemolyticus based on prokaryotic Argonaute protein, aiming to solve many problems that still exist in CRISPR / Cas technology, such as the high cost and instability of guide RNA, the inherent off-target nature of Cas effector protein, and the targeting mechanism that strictly relies on the protospacer sequence adjacent motif, which largely limits the application scope of LAMP detection of Vibrio parahaemolyticus.

[0005] The first technical solution provided by the present invention is a composition, which includes a pair of gDNA (gF, gT) and a probe, wherein the gDNA is selected from any one of the groups 1) to 8): 1) gDNA gF1, gDNA gT1; 2) gDNA gF2, gDNA gT2; 3) gDNA gF3, gDNA gT3; 4) gDNA gF4, gDNA gT4; 5) gDNA gF5, gDNA gT5; 6) gDNA gF6, gDNAgT6; 7) gDNA gF7, gDNA gT7; 8) gDNA gF8, gDNA gT8; the probe is selected from any one of the groups 9) to 10): 9) probe Pb1; 10) probe Pb2. wherein the gDNA gF1 nucleotide sequence is as shown in SEQ ID NO:5; the gDNA gT1 nucleotide sequence is as shown in SEQ ID NO:6; the gDNA gF2 nucleotide sequence is as shown in SEQ ID NO:7; the gDNA gT2 nucleotide sequence is as shown in SEQ ID NO:8; the gDNA gF3 nucleotide sequence is as shown in SEQ ID NO:9; the gDNAgT3 nucleotide sequence is as shown in SEQ ID NO:10; the gDNA gF4 nucleotide sequence is as shown in SEQ ID NO:11; the gDNA gT4 nucleotide sequence is as shown in SEQ ID NO:12; the probe Pb1 nucleotide sequence is as shown in SEQ ID NO:13; the gDNA gF5 nucleotide sequence is as shown in SEQ ID NO:14; the gDNA gT5 nucleotide sequence is as shown in SEQ ID NO:15; the gDNA gF6 nucleotide sequence is as shown in SEQ ID NO:16; the gDNA gT6 nucleotide sequence is as shown in SEQ ID NO:17; the gDNA gF7 nucleotide sequence is as shown in SEQ ID NO:18; the gDNA gT7 nucleotide sequence is as shown in SEQ ID NO:19; the gDNA gF8 nucleotide sequence is shown in SEQ ID NO:20; the gDNA gT8 nucleotide sequence is shown in SEQ ID NO:21; the probe Pb2 nucleotide sequence is shown in SEQ ID NO:22.

[0006] In certain embodiments, the 5' end of the gDNA is phosphorylated.

[0007] In certain embodiments, 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.

[0008] In certain embodiments, the fluorescent reporter group is selected from FAM, HEX, JOE, CY3, and CY5, and the quencher group is selected from DABCYL, TAMRA, and BHQ.

[0009] Preferably, the 5' end of the probe is modified with a fluorescent reporter group FAM.

[0010] The second technical solution provided by the present invention is a kit for detecting Vibrio parahaemolyticus, wherein the kit comprises the composition described in the first technical solution and a LAMP amplification composition.

[0011] In certain embodiments, the LAMP amplification system composition comprises a pair of external primers F3 and B3 and a pair of internal primers FIP and BIP; the nucleic acid sequence of the external primer F3 is shown in SEQ ID NO:1, the nucleic acid sequence of the external primer B3 is shown in SEQ ID NO:2, the nucleic acid sequence of the internal primer FIP is shown in SEQ ID NO:3, and the nucleic acid sequence of the internal primer BIP is shown in SEQ ID NO:4.

[0012] In certain embodiments, the kit further comprises a LAMP amplification reagent and Pf Ago detection reagents.

[0013] In certain embodiments, the LAMP amplification reagent includes 10×Isothermal Amplification Buffer II, MgSO 4 , dNTP Mix and Bst 3.0 DNA Polymerase.

[0014] In certain embodiments, the Pf Ago detection reagents include MnCl2, 10× Pf Ago Endonuclease Reaction Buffer and Pf Ago Endonuclease.

[0015] In certain embodiments, the kit further comprises a positive control substance for Vibrio parahaemolyticus.

[0016] The third technical solution provided by the present invention is a method for detecting Vibrio parahaemolyticus for non-disease diagnosis purposes, wherein the method is to detect Vibrio parahaemolyticus using the kit described in the second technical solution.

[0017] In certain embodiments, the method comprises the steps of:

[0018] (1) Extract genomic DNA from the sample to be tested or directly dilute the sample to be tested;

[0019] (2) constructing a LAMP amplification system using the LAMP amplification composition to perform LAMP amplification;

[0020] (3) The LAMP amplification product obtained in step (2) is constructed using the composition described in the first technical solution. Pf Ago detection system, enzyme digestion and fluorescence detection.

[0021] In certain embodiments, the total volume of the LAMP amplification system is 25 μL, 10×IsothermalAmplification Buffer II 2.5 μL, MgSO4 (100 mM) 1 μL, dNTP Mix (10 mM) 3.5 μL, inner primers FIP and BIP (10 μM) are added 3 μL respectively, outer primers F3 and B3 (10 μM) are added 0.5 μL respectively, Bst 3.0 DNA Polymerase (8000 U / mL) is added 0.75 μL, and the nucleic acid to be tested is 2 μL, and ddH2O is added to make up to 25 μL; the amplification program is 69°C, 30 min.

[0022] In certain embodiments, the Pf The total volume of the Ago detection system was 20 μL, 2 μL of gDNA gF5 and gT5 (20 μM) were added respectively, 1 μL of probe Pb2 (20 μM), 0.5 μL of MnCl2 (40 mM), and 10× Pf Ago EndonucleaseReaction Buffer 2 μL, Pf Ago Endonuclease (200 U / μL) 2 μL, LAMP product 4 μL, add ddH2O to make up to 20 μL; fluorescence detection conditions are 95 ℃, 15 min.

[0023] The fourth technical solution provided by the present invention is the use of the composition described in the first technical solution in the preparation of a product for detecting Vibrio parahaemolyticus.

[0024] The present invention has the following beneficial effects:

[0025] (1) The present invention effectively combines LAMP amplification and Pf The advantages of Ago cleavage were used to establish a LAMP-based assay for Vibrio parahaemolyticus. Pf Ago rapid detection method has the advantages of simple operation and low equipment requirements. Compared with the simple LAMP technology, PfAgo can target and cut complementary single-stranded DNA and is less affected by the environment, which effectively reduces false positives and thus expands the application scenarios. Pf Ago has the advantages of not relying on the adjacent motif of the pre-spacer sequence and being more stable.

[0026] (2) The present invention designs multiple groups of gDNA and screens gDNA and fluorescent probes with high cutting efficiency. Among the 28 experimental strains, only the positive strain of Vibrio parahaemolyticus showed amplification signals, while other Vibrio (Vibrio alginolyticus, Vibrio vulnificus, Vibrio cholerae) and negative control bacteria of non-Vibrio genus showed no amplification phenomenon.

[0027] (3) This invention is the first to use LAMP- Pf Ago technology was applied to the detection of Vibrio parahaemolyticus, and the detection could be completed within 45 min. The method had high sensitivity, and the detection limit of pure culture was 5.47 CFU / mL. In the artificial contamination test, the minimum detectable initial inoculum was 7.84×10 1 The target bacteria of CFU / mL have good practical application effects. In addition, the present invention can directly visually interpret the results by observing the color change of the reaction product under ultraviolet light, which can meet the detection needs of both on-site and laboratory. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Optimization of LAMP reaction conditions; A: Reaction temperature optimization; B: Internal primer concentration optimization; C: Mg 2+ concentration optimization; D: dNTPs concentration optimization; E: Bst 3.0 DNA polymerase concentration optimization.

[0029] Figure 2 The cleavage effects of different gDNAs on LAMP amplification products.

[0030] Figure 3 for Pf Optimization of Ago cutting conditions; A: Optimization of LAMP amplification product dosage; B: Pf Ago enzyme concentration optimization; C: gDNAs concentration optimization; D: MnCl2 concentration optimization; E: fluorescent probe concentration optimization.

[0031] Figure 4 For LAMP- Pf Ago method specific detection results.

[0032] Figure 5 For LAMP- Pf Sensitivity evaluation results of the Ago method; A: fluorescence intensity of detection at different bacterial concentrations; B: UV detection results at different bacterial concentrations, where tube numbers 1 to 8 are 5.47×106 , 5.47×10 5 , 5.47×10 4 , 5.47×10 3 , 5.47×10 2 , 5.47×10 1 , 5.47, 0 CFU / mL. DETAILED DESCRIPTION

[0033] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0034] Materials used in the examples:

[0035] The main reagents and culture medium used in the examples: 10×Isothermal Amplification Buffer II, MgSO4 (100 mM), Bst 3.0 DNA Polymerase were purchased from New England Biolabs; dNTPs Mix (10 mM each) (Beijing Solebow Technology Co., Ltd.); Syto 9 Green fluorescent dye (Invitrogen); 10× Pf Ago Endonuclease Reaction Buffer, MnCl2 (40 mM), Pf Ago Endonuclease (200 U / μL) was purchased from Beijing Baoying Tonghui Biotechnology Co., Ltd.; 3% sodium chloride alkaline peptone water (APW) and LB broth were purchased from Beijing Luqiao Technology Co., Ltd.

[0036] The main instruments and equipment used in the examples are: KB240 low-temperature incubator (Binder, Germany); TS-200B constant temperature shaker (Shanghai Tiancheng Experimental Instrument Manufacturing Co., Ltd.); 1-14 desktop centrifuge (Sigma, Germany); Bagmixer400 slapping homogenizer (Interscience, France); 1300 series A2 biological safety cabinet (Thermo Fisher Scientific, USA); CFX96 real-time fluorescence quantitative PCR instrument (Bio-Rad, USA).

[0037] Example 1: Establishment of LAMP detection method

[0038] 1.1 LAMP primer design

[0039] Based on the conserved region of the specific target gene of Vibrio parahaemolyticus, LAMP primers were designed using the online software PrimerExplorer V5. All primers were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The specific primer sequences are as follows:

[0040] External primer F3: 5′-TCTTGGTGAGGAAATCATGCA-3′ (SEQ ID NO: 1);

[0041] External primer B3: 5′-CCTTACTGTTCCCATTCTTCTCG-3′ (SEQ ID NO: 2);

[0042] Inner Primer FIP:

[0043] 5'-TTCCTGGCGTTCCGTTGGTGACAAACGTCGCTCTGGATG-3' (SEQ ID NO: 3);

[0044] Inner Primer BiP:

[0045] 5'-GGTCGCGGCACGACCATTTTGAAGGGCAAACCATCGGC-3' (SEQ ID NO: 4).

[0046] 1.2 Reaction conditions and system optimization

[0047] The reaction temperature, inner primer concentration, Mg 2+ The parameters such as concentration, dNTPs concentration, and amount of Bst 3.0 DNA polymerase added were optimized. In addition, the saturated fluorescent dye Syto 9 Green was added to achieve real-time monitoring of the entire reaction process.

[0048] Reaction temperature optimization: The initial temperature was 64.8 °C, and the experimental setting temperatures were 62.5 °C, 64.8 °C, 67.9 °C, and 69 °C. The optimal reaction temperature was determined by fluorescence curve analysis. Figure 1 As shown in A in the figure, the peak time of the fluorescence curve is earlier at 64.8 ℃, 67.9 ℃, and 69 ℃, and the peak time of the fluorescence curve is the latest at 62.5 ℃. In order to ensure the specificity of the amplified product, 69 ℃ was selected as the optimal reaction temperature for LAMP detection.

[0049] Optimization of inner primer concentration: Keep the outer primer concentration unchanged and set the inner primer concentration to 0.8 µM, 1.2 µM, 1.6 µM, and 2.0 µM. Figure 1As shown in Figure B, there is no significant difference in the peak time of the fluorescence curve between different concentrations of internal primers (FIP / BIP). Compared with the final concentrations of internal primers of 0.8 µM, 1.6 µM, and 2.0 µM, the peak time was slightly earlier at 1.2 µM, so 1.2 µM was selected as the optimal internal primer concentration for LAMP detection.

[0050] Mg 2+ Concentration optimization: Setting Mg 2+ The concentrations are 4 mM, 6 mM, 8 mM, and 10 mM. Figure 1 It can be seen from the C in that as Mg 2+ As the concentration increases, the peak time of the fluorescence curve gradually increases, indicating that too much Mg 2+ will inhibit the amplification efficiency of the LAMP reaction, therefore, 4 mM Mg 2+ It is the optimal concentration for LAMP detection system.

[0051] Optimization of dNTPs concentration: set the dNTPs concentration to 1.0 mM, 1.4 mM, 1.8 mM, and 2.0 mM. Figure 1 D in the figure shows that when the dNTPs concentration is less than 1.4 mM or greater than 1.8 mM, the peak time of the fluorescence curve is delayed, indicating that the LAMP reaction is inhibited to varying degrees. When the dNTPs concentration is 1.4 mM, the peak time is the earliest, so 1.4 mM is selected as the optimal dNTPs concentration for LAMP detection.

[0052] Optimization of Bst 3.0 DNA polymerase addition amount: Set the Bst 3.0 DNA polymerase addition amount to 0.5 µL (160 U / mL), 0.75 µL (240 U / mL), 1 µL (320 U / mL), and 1.25 µL (400 U / mL). Figure 1 As shown in Figure E, when the concentration of BstDNA polymerase is 240-400 U / mL, the peak time of the fluorescence curve is close and the curve is smooth, while when the concentration is less than 240 U / mL, the peak time is significantly delayed. Considering the amplification efficiency and economic cost, 240 U / mL is selected as the optimal Bst DNA polymerase concentration for LAMP detection.

[0053] 1.3 Determining the optimal conditions for LAMP detection

[0054] The reaction system was as follows: 2.5 μL of 10×Isothermal Amplification Buffer II, 1 μL of MgSO4 (100 mM), 3.5 μL of dNTP Mix (10 mM), 3 μL of inner primers FIP and BIP (10 μM), 0.5 μL of outer primers F3 and B3 (10 μM), 0.75 μL of Bst 3.0 DNA Polymerase (8000 U / mL), 2 μL of nucleic acid to be tested, and ddH2O was added to make up to 25 μL. The reaction conditions were 69 °C for 30 min.

[0055] Embodiment 2: Pf Establishment of Ago cleavage method

[0056] 2.1 Screening of gDNAs

[0057] 8 pairs of gDNAs (gF and gT) were designed for LAMP amplification products. Pf Ago cuts one single strand of the double-stranded DNA to obtain a single-stranded DNA with a 5' phosphate group, which is the newly generated gDNA (gN). Pf Ago targeted cleavage of complementary fluorescent probes, and the fluorescent signal generated by the reaction was detected. Among them, the fluorescent probe Pb1 was used in combination with gF1-gT1, gF2-gT2, gF3-gT3, and gF4-gT4; the fluorescent probe P2 was used in combination with gF5-gT5, gF6-gT6, gF7-gT7, and gF8-gT8. The gDNAs and fluorescent probes used were synthesized by Nanjing GenScript Biotechnology Co., Ltd., as shown in Table 1.

[0058] Table 1 gDNAs and fluorescent probes for detecting Vibrio parahaemolyticus

[0059]

[0060] First, the eight pairs of gDNAs were Pf Ago cleavage reaction, taking into account the fluorescence generation rate and fluorescence intensity, to determine the best gDNAs. Figure 2 As shown, different gDNAs mediate Pf The rate of fluorescence signal generation by Ago cleavage reaction varies greatly, among which gF5 / gT5, gF8 / gT8 and gF1 / gT1 can reach the highest fluorescence intensity within 10 min. It can be seen that gF5 / gT5 has higher cleavage efficiency and fluorescence intensity, so gF5 / gT5 is selected as the best gDNAs.

[0061] 2.2 Reaction system optimization

[0062] Add 10 U / µL, 20 U / µL, 40 U / µL, 80 U / µL of the final concentration into the 20 µL reaction system. Pf Ago, MnCl2 at a final concentration of 0.5 mM, 1 mM, 2 mM, 4 mM, gDNAs at a final concentration of 0.5 µM, 1 µM, 2 µM, 4 µM, fluorescent probes at a final concentration of 0.5 µM, 1 µM, 2 µM, 3 µM, and finally 2 µL of LAMP product was added and mixed. The reaction was performed at 95 °C for 15 min, during which 15 cycles were set up, and the fluorescence value was monitored for each cycle (minute).

[0063] Optimization of the amount of LAMP amplification product: Figure 3 As shown in A, when the amount of amplification product was 1 μL and 2 μL, the fluorescence signal values ​​of both were high; in contrast, the peak time of the 2 μL amplification product was shorter, and it entered the plateau phase after 10 min of reaction. When the amount of amplification product was increased to 3 μL and 4 μL, the peak time was shortened, but the fluorescence signal value decreased. Considering the fluorescence signal value and peak time, the amount of LAMP amplification product was determined to be 4 μL.

[0064] Pf Optimization of Ago enzyme addition: Figure 3 From B in the figure, we can see that when Pf When the Ago enzyme concentration was 10 U / μL and 20 U / μL, there was no significant difference in the peak time between the two. Pf When the Ago enzyme concentration was 20 U / μL, the fluorescence signal value was higher and entered the plateau phase after 10 min of reaction. Pf When the Ago enzyme concentration increased to 40 U / μL and 80 U / μL, the peak time was slightly prolonged and the fluorescence signal value decreased. Therefore, 20 U / μL was selected as Pf Optimal enzyme concentration for Ago cleavage reaction.

[0065] Optimization of gDNA concentration: Figure 3 As shown in C, as the amount of gDNAs added increases, the reaction peak time gradually shortens. When the gDNAs concentration is 1 μM and 2 μM, it can enter the plateau phase within 10 min; in contrast, the fluorescence signal value of the 2 μM added concentration is higher, so 2 μM is selected as Pf Optimal gDNA concentration for Ago cleavage reaction.

[0066] MnCl2 concentration optimization: Figure 3 As shown in D, as the concentration of MnCl2 increases, the peak time of the fluorescence curve gradually increases, indicating that too much MnCl2 cannot improve PfAgo cleavage reaction efficiency. When the MnCl2 concentration is 1 mM, the reaction peak time is the earliest, so 1 mM MnCl2 is selected as the optimal concentration of the cleavage system.

[0067] Optimization of fluorescent probe addition: Figure 3 As shown in E, with the increase of the amount of probe added, the fluorescence signal value gradually increased, but the peak time was slightly prolonged. The peak time of 0.5 μM and 1 μM additions was shorter, and the reaction entered the plateau phase within 10 min. Considering the fluorescence signal intensity and detection cost, 1 μM fluorescent probe was selected as the optimal concentration of the cleavage system.

[0068] 2.3 Determination Pf Optimal conditions for Ago cleavage

[0069] The reaction system was as follows: 2 μL of gDNA gF5 and gT5 (20 μM), 1 μL of probe Pb2 (20 μM), 0.5 μL of MnCl2 (40 mM), 10× Pf Ago Endonuclease Reaction Buffer 2 μL, Pf 2 μL of Ago Endonuclease (200 U / μL), 4 μL of LAMP product, add ddH2O to make up to 20 μL. The reaction conditions are 95 ℃, 15 min.

[0070] Example 3: LAMP- Pf Ago specificity validation

[0071] The genomic DNA of 16 positive strains of Vibrio parahaemolyticus and 12 negative strains of non-Vibrio parahaemolyticus (Table 2) preserved in our laboratory was used as template and the LAMP- Pf Ago method was used for detection.

[0072] Table 2 Strains used for specificity evaluation

[0073] Strain name Latin name Strain number Vibrio parahaemolyticus ATCC 33847 Vibrio parahaemolyticus ATCC 17802 Vibrio parahaemolyticus NJIFDCVp1-14 Vibrio alginolyticus ATCC 17749 Vibrio alginolyticus ATCC 33787 Vibrio vulnificus ATCC 27562 Vibrio vulnificus CICC 25009 Vibrio cholerae CICC 23794 Vibrio cholerae CICC 23795 Salmonella Typhimurium Typhimurium ATCC 14028 Escherichia coli ATCC 25922 Staphylococcus aureus CMCC(B)260031 Listeria monocytogenes ATCC 19115 Pseudomonas aeruginosa ATCC 9027 Bacillus cereus CMCC(B)63303

[0074] Note: ATCC (American Type Culture Collection), American Type Culture Collection; CICC (China Center of Industrial Culture Collection), China Center for Industrial Microbiology Collection; CMCC (China Medical Culture Collection), China Center for Medical Bacteria Collection; NJIFDCVp1-14 indicates 14 strains of Vibrio parahaemolyticus isolates.

[0075] Method-specific test results such as Figure 4 As shown in the figure, all 16 positive strains of Vibrio parahaemolyticus showed amplification curves, while the other 12 negative strains did not produce fluorescence curves, indicating that the constructed LAMP- Pf The Ago method has good specificity.

[0076] Example 4: LAMP- Pf Ago Sensitivity Verification

[0077] Take the bacterial solution cultured to the logarithmic growth phase and dilute it 10 times to 10 with sterile saline. -8 Each gradient dilution was boiled to extract DNA, and used as a template for LAMP- Pf Ago detection is used 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.

[0078] In LAMP- Pf In the sensitivity evaluation of the Ago method, the concentration of Vibrio parahaemolyticus was 5.47×10 7 CFU / mL, the concentration obtained by gradient dilution was 5.47×10 6 ~5.47 CFU / mL of bacterial solution. Figure 5 As shown in A, compared with the blank control, the bacterial concentration that produced the lowest fluorescent signal was 5.47 CFU / mL. At the same time, the reaction results were also observed by ultraviolet light ( Figure 5 In B), the positive reaction emits green fluorescence under ultraviolet irradiation, while the blank control does not emit fluorescence. It can be found that this method can clearly distinguish positive samples from negative samples and can be used for the qualitative detection of Vibrio parahaemolyticus.

[0079] Example 5: Detection of artificially contaminated samples

[0080] Prepare salmon that has tested negative for Vibrio parahaemolyticus according to GB 4789.7-2013 "National Food Safety Standard - Microbiological Examination of Foods - Vibrio parahaemolyticus", weigh 25 g of the sample, and then add 10 2 ~10 8Prepare artificial contaminated samples by adding 2.5 mL of CFU / mL suspension of Vibrio parahaemolyticus. Add 225 mL of 3% sodium chloride alkaline peptone water to the artificial contaminated samples, homogenize to prepare a 1:10 sample solution (the bacterial solution dilution ratio is 1:100 at this time), and shake culture at 36 ℃±1 ℃ for 0, 2, and 4 h. At the same time, add sterile water to the sample as a negative control. Pipette 1 mL of the homogenate into a 1.5 mL centrifuge tube, wash twice with sterile water, shake and mix, and then perform LAMP- Pf Ago detection.

[0081] As shown in Table 3, without culture, LAMP- Pf The minimum initial inoculum size that can be detected by the Ago method is 7.84×10 1 CFU / mL of target bacteria. After 2 h of culture, the initial inoculum size was 7.84×10 0 CFU / mL salmon samples can be detected. At the same time, it can be seen that the blank controls with culture time of 0, 2, and 4 h did not detect the target bacteria. In addition, without pre-enrichment, 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 caused by various matrices in the actual sample. Therefore, in the actual sample detection, the appropriate pre-enrichment step can be selected according to the time requirements.

[0082] Table 3 Test results of artificially contaminated samples

[0083]

[0084] Note: “+” indicates detected; “-” indicates not detected.

[0085] Although the present invention has been disclosed as above in the form of a preferred embodiment, 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 kit for detecting Vibrio parahaemolyticus, characterized in that: The kit includes a LAMP amplification composition and Pf Ago cleavage composition, the LAMP amplification composition includes a pair of external primers F3 and B3 and a pair of internal primers FIP and BIP; the nucleic acid sequence of the external primer F3 is shown in SEQ ID NO: 1, the nucleic acid sequence of the external primer B3 is shown in SEQ ID NO: 2, the nucleic acid sequence of the internal primer FIP is shown in SEQ ID NO: 3, the nucleic acid sequence of the internal primer BIP is shown in SEQ ID NO: 4, the Pf The Ago cleavage composition includes a pair of guide DNA gF5 and gT5 and a probe Pb2; the nucleic acid sequence of the gDNA gF5 is shown in SEQ ID NO: 14, the nucleic acid sequence of the gDNA gT5 is shown in SEQ ID NO: 15, and the nucleic acid sequence of the probe Pb2 is shown in SEQ ID NO: 22; The kit also includes a LAMP amplification reagent and Pf Ago detection reagent; The LAMP amplification reagent includes 10× isothermal amplification buffer II, MgSO4, dNTP Mix and Bst 3.0 DNA polymerase; Pf Ago detection reagents include MnCl2, 10× Pf Ago endonuclease reaction buffer and Pf Ago endonuclease; The LAMP amplification composition and the LAMP amplification reagent constitute a LAMP amplification system, wherein the total volume of the LAMP amplification system is 25 μL, 10× isothermal amplification buffer II 2.5 μL, 100 mM MgSO4 1 μL, 10 mM dNTP Mix 3.5 μL, 10 μM inner primers FIP and BIP are added 3 μL respectively, 10 μM outer primers F3 and B3 are added 0.5 μL respectively, 8000U / mL Bst 3.0 DNA polymerase 0.75 μL, and the nucleic acid to be detected is 2 μL, and ddH2O is added to make up to 25 μL; Said Pf Ago cleavage composition and the Pf Ago detection reagent composition Pf Ago detection system, Pf The total volume of the Ago detection system was 20 μL, 2 μL of 20 μM gDNA gF5 and gT5 were added, 1 μL of 20 μM probe Pb2, 0.5 μL of 40 mM MnCl2, and 10× Pf Ago endonuclease reaction buffer 2 μL, 200 U / μL Pf 2 μL of Ago endonuclease and 4 μL of LAMP product were added to 20 μL with ddH2O.

2. The kit according to claim 1, characterized in that The 5' end of the gDNA is phosphorylated.

3. The kit according to claim 1, characterized in that 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.

4. The kit according to claim 1, characterized in that The kit also contains a positive control substance of Vibrio parahaemolyticus.

5. A method for detecting Vibrio parahaemolyticus for non-disease diagnosis purposes, characterized in that: The method comprises the step of using the kit described in any one of claims 1 to 4 to detect Vibrio parahaemolyticus.

6. The method according to claim 5, 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 a LAMP amplification system using the LAMP amplification composition to perform LAMP amplification; (3) The LAMP amplification product obtained in step (2) is used Pf Ago cleavage composition construction Pf Ago detection system, enzyme digestion and fluorescence detection.

7. The method according to claim 6, characterized in that The total volume of the LAMP amplification system is 25 μL, 10× isothermal amplification buffer II 2.5 μL, 100 mM MgSO4 1 μL, 10 mM dNTP Mix 3.5 μL, 10 μM inner primers FIP and BIP 3 μL, 10 μM outer primers F3 and B3 0.5 μL, 8000 U / mL Bst 3.0 DNA polymerase 0.75 μL, 2 μL of nucleic acid to be tested, and ddH2O is added to make up to 25 μL; the amplification program is 69 ° C, 30 min; Said Pf The total volume of the Ago detection system was 20 μL, 2 μL of 20 μM gDNA gF5 and gT5 were added, 1 μL of 20 μM probe Pb2, 0.5 μL of 40 mM MnCl2, and 10× Pf Ago endonuclease reaction buffer 2 μL, 200 U / μL Pf 2 μL of Ago endonuclease and 4 μL of LAMP product were added with ddH2O to make up to 20 μL; the fluorescence detection conditions were 95 ℃ for 15 min.

Images