Method for detecting PRV (pseudorabies virus) by PCR-ELISA (polymerase chain reaction-enzyme linked immunosorbent assay)
A technology of PRV-F and PRV-R, which is applied in the field of detecting PRV by PCR-ELISA, can solve the problems of high experimental cost, poor sensitivity, and low sensitivity of ordinary PCR gel electrophoresis, and eliminate the complicated ELISA sample preparation and improve Specificity and sensitivity, and the effect of reducing the difficulty of the test
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Embodiment 1
[0040] Example 1 Primer Design
[0041]According to the GenBank swine fever Shimen strain sequence (accession number: JQ809329.1), design specific primers PRV-F, PRV-R for general PCR amplification, the gene fragment is 399bp, and then respectively at the 5' end of the upstream and downstream Digoxigenin (DIG) and biotin (BIO) were labeled respectively, and a pair of specific primers PRV-IF and PRV-IR were designed for fluorescent quantitative PCR amplification. The gene length was 125bp, and the primers were provided by Shanghai Sangon Bioengineering AGs were synthesized and labeled as shown in Table 1.
[0042] Table 1 Primers
[0043]
Embodiment 2
[0044] Embodiment 2 PRV ordinary PCR amplification
[0045] Take out the porcine pseudorabies virus tissue disease material preserved in the laboratory from the -80°C refrigerator, take about 10 mg of lymph nodes, and extract viral DNA according to the instructions of TaKaRa MiniBEST ViralRNA / DNAExtraction Kit Ver.5.0. PCR amplification was carried out with PRV DNA as template and PRV-F and PRV-R as primers. The PCR reaction conditions were pre-denaturation at 94°C for 4 minutes; 30 cycles at 94°C for 30s, 56°C for 30s, and 72°C for 30s; extension at 72°C for 5 minutes; after the end, 5 μL of the PCR product was taken for agarose gel electrophoresis, and the results were as follows: figure 1 As shown, the target band with a size of about 399bp was amplified, which was consistent with the expected target fragment size.
Embodiment 3
[0046] Construction of embodiment 3 positive plasmid
[0047] The above PCR product was connected to the cloning vector PMD19-T Vector. The connection system was PMD19-T Vector 0.5 μL, target gene 4.5 μL, Solution I 5.0 μL, the total volume was 10.0 μL, mixed well and placed in a constant temperature bath at 16°C overnight for connection. The next day, take out Escherichia coli DH5α competent cells from the -80°C refrigerator and place them in a mixture of ice and water to dissolve them, add 5 μL of the ligation product, bathe in ice for 30 minutes, heat shock at 42°C for 90 seconds, take them out immediately, place them on ice for 1-2 minutes, add 900 μL without Put the resistant LB liquid medium in a 37°C incubator for 45 minutes, centrifuge at 4000rpm for 5 minutes, keep about 100 μL of supernatant, and evenly spread it on the LB solid medium plate containing Amp resistance (100 μg / mL). Place the plate upside down in a constant temperature incubator at 37°C for overnight c...
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