[0039] Example
[0040] A kit for detecting Vibrio parahaemolyticus in aquatic products, including sterile deionized water, PCR reaction solution, Taq DNA polymerase, ethidium bromide DNA dye, bromophenol blue loading buffer, standard substance, reference substance;
[0041] Among them, the PCR reaction solution contains PCR reaction buffer, MgCl 2 , Deoxyribonucleoside triphosphates (dNTPs), primers for detection;
[0042] The detection primers are a mixture of the following 4 primers (Table 1).
[0043] Table 1
[0044]
[0045] The detection primers used the D-type amino acid dehydrogenase small subunit gene of Vibrio parahaemolyticus as template DNA, designed with Primer Premier 5, and synthesized by Shenggong Bioengineering (Shanghai) Co., Ltd.;
[0046] The standard product is a recombinant plasmid containing the amplified product obtained after PCR amplification using the Vibrio parahaemolyticus genome as template DNA and P3 and P4 as primers, which is constructed by the following method: clone the amplified product into pMD 18-T The vector is transformed into E. coli TOP10 competent cells. The PCR-positive recombinant plasmid is sequenced to verify that the target gene is correct; P3 and P4 are primers and the amplified product sequence is shown in SEQ ID No. 6 in the sequence table;
[0047] The control substance is divided into a negative control substance and a positive control substance. The negative control substance is sterile deionized water, and the positive control substance is a DNA fragment obtained by gel electrophoresis purification of amplified products with P3 and P4 primers. The sequence is shown in the preface List SEQ ID No. 6.
[0048] The kit is stored at -20°C in the dark to avoid repeated freezing and thawing.
[0049] Collect 3 groups of small yellow croaker, 2 groups of sea rainbow, 1 group of oysters, 2 groups of clams, 2 groups of razor clams, 2 groups of green prawns, 1 group of crabs, and 1 group of snails from different aquatic product markets. A total of 14 groups of aquatic samples were divided into two. (As the test sample of the kit of the present invention and the test sample of the national standard method), each portion is not less than 50g, respectively as sample 5 to sample 18. 3 groups of small yellow croaker correspond to sample 5, sample 6, sample 7, and 2 groups of Haihong Corresponding to sample 8 and sample 9, one group of oysters corresponds to sample 10, two groups of clams correspond to sample 11 and sample 12, two groups of clams correspond to sample 13 and sample 14, and two groups of clams correspond to sample 15 and sample 16, 1 Group crabs correspond to sample 17, and group 1 snails correspond to sample 18. They are used immediately for testing or stored at -20℃.
[0050] Use the kit of the present invention to detect Vibrio parahaemolyticus in 14 groups of aquatic samples:
[0051] Take 9 g of samples 5~18 aseptically according to the type of sample (take the intestines and gills for fish, take the hepatopancreas and digestive caecum from the cephalothorax for shrimps, take the viscera and gill strips for crabs, and take belly for snails) In the lower part of the foot muscles, the shellfish are taken from the internal organs and gills of the outer layer of the axe foot muscles, respectively, and homogenized in a sterile mortar or tissue grinder. Add 50 ml of sterile 3% sodium chloride alkaline peptone water (Peptone 10g, sodium chloride 30g, distilled water 1000ml, pH 8.5, autoclaved at 121°C for 15min after aliquoting), incubate at 28°C for 8h-12h (sample 5~9 for 8h, sample 10~14 for 10h , Sample 15~Sample 18 culture for 12h).
[0052] (B) DNA extraction: take the cultured enrichment solution, shake and mix, centrifuge at 2000 rpm for 5 min, take the supernatant and centrifuge at 10,000 rpm for 10 min, discard the supernatant, resuspend the pellet in sterile saline, and centrifuge at 10,000 rpm for 10 Min, discard the supernatant, resuspend the pellet in physiological saline, boil water bath for 10 min, ice bath for 5 min, centrifuge at 10,000 rpm for 10 min, and take the supernatant as the sample template DNA.
[0053] (C) Preparation of PCR reaction tube: The preparation of PCR reaction tube and the melting of PCR reaction solution should be carried out on ice. Take 27.8μl of PCR reaction solution in turn, Taq 0.2 μl of DNA polymerase and 2 μl of sample template DNA were added to the PCR reaction tube and mixed uniformly to prepare a PCR reaction tube of sample template DNA. Take 27.8μl of PCR reaction solution, Taq 0.2μl of DNA polymerase and 2μl of sterile deionized water were added to the PCR reaction tube and mixed evenly to prepare a PCR reaction tube of negative control substance. Take 27.8μl of PCR reaction solution, aq DNA polymerase 0.2μl, P3 and P4 as primers of the amplified products are purified by gel electrophoresis, and 2μl of DNA fragments obtained are added to the PCR reaction tube and mixed uniformly to prepare the PCR reaction tube of the positive control substance. Take 27.8μl of PCR reaction solution, aq 0.2μl of DNA polymerase, 2μl of recombinant plasmid containing the amplified product obtained after PCR amplification using Vibrio parahaemolyticus genome as template DNA and primers P3 and P4 were added to the PCR reaction tube and mixed evenly to prepare a standard product PCR reaction tube. (A positive control and a negative control should be set up for each test).
[0054] (D) Amplification detection: Place the PCR reaction tube prepared in step (d) on the PCR machine, and perform amplification according to the following reaction procedure: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 1 min, 59.6℃ annealing for 1 min, 72℃ Extend for 75s, 30 cycles in total; extend at 72°C for 8 min; denature at 94°C for 1 min, annealing at 56°C for 1 min, and extend at 72°C for 30s, total 30 cycles; extend at 72°C for 8 min to obtain PCR amplification products (store at 4°C Or immediately used to determine the test results).
[0055] (E) Judgment of test results: Dissolve 0.3g agarose in 30ml electrophoresis buffer, mix well and boil it, add ethidium bromide with a final concentration of 0.5μg/ml when it is cooled to about 60℃, and pour the gel plate. After the gel is cooled and solidified, 3μl of the PCR amplification product is mixed with the same amount of loading buffer, and added to the gel well for electrophoresis, and electrophoresis is carried out at 5V/cm for 40 min. After the electrophoresis is over, take out the gel and place it under ultraviolet light for observation and photographing. The standard gel wells and the positive control gel wells have electrophoresis bands, but the negative control gel wells have no bands, indicating that the PCR reaction is successful. The gel hole of the tested sample (such as Figure 4 Electrophoresis bands and standard gel wells (such as image 3 with Figure 4 Middle 3 lanes), positive control gel wells (such as image 3 with Figure 4 Middle 4 lanes) If the corresponding band size is the same, it is judged as positive, otherwise, it is judged as negative. Figure 4 Shown. by Figure 4 It can be seen that the test samples in lanes 5, 7, 9, 11, 15, 17 are negative for Vibrio parahaemolyticus, and the samples in lanes 6, 8, 10, 12, 13, 14, 16, and 18 are negative for parahaemolytic arcs. Bacteria is positive, indicating that sample 5, sample 7, sample 9, sample 11, sample 15, and sample 17 are negative for Vibrio parahaemolyticus, sample 6, sample 8, sample 10, sample 12, sample 13, sample 14, sample 16, Sample 18 is positive for Vibrio hemolyticus.
[0056] The 14 groups of aquatic samples were tested for Vibrio parahaemolyticus, and were tested in accordance with the national standard (GB/T 4789.7-2008) "Food Hygiene Microbiological Examination of Vibrio parahaemolyticus" as a control method for comparison with the present invention . According to national standard methods, sample 5, sample 7, sample 9, sample 11, sample 15, sample 17 are negative for Vibrio parahaemolyticus, sample 6, sample 8, sample 10, sample 12, sample 13, sample 14, sample 16, Sample 18 is positive for Vibrio hemolyticus.
