Method for detecting CSFV by utilizing PCR-ELISA
A technique of CSFV-F and CSFV-R, which is applied in the field of detection of CSFV 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: AY775178.2), specific primers CSFV-F and CSFV-R were designed for ordinary PCR amplification. The gene fragment was 511 bp; then the 5'ends of the primers were respectively located upstream and downstream Labeling digoxin (DIG) and biotin (BIO) separately; design a pair of specific primers CSFV-IF and CSFV-IR for fluorescence quantitative PCR amplification, the gene length is 114bp; the primers are provided by Shanghai Shenggong Bioengineering Co., Ltd. synthesizes and signs, as shown in Table 1.
[0042] Table 1 Primers
[0043]
Embodiment 2
[0044] Example 2 CSFV ordinary PCR amplification
[0045] Take out the swine fever virus tissue disease material stored in the laboratory from the refrigerator at -80°C, take about 10 mg of lymph nodes, extract viral RNA according to TaKaRaMiniBEST ViralRNA / DNA Extraction Kit Ver.5.0 instructions, and then reverse transcribe cDNA. Using CSFV cDNA as template and CSFV-F and CSFV-R as primers for PCR amplification. The PCR reaction system and conditions were 94°C pre-denaturation 4min; 94°C 30s, 55°C 30s, 72°C 40s, 30 cycles; 72°C extension for 5min; after the end, take 5μL of PCR product for agarose gel electrophoresis, the results are as follows figure 1 As shown, a target band with a size of about 511 bp was amplified, which was consistent with the expected target fragment size.
Embodiment 3
[0046] Example 3 Construction of positive plasmid
[0047] Connect the above PCR product to the cloning vector PMD19-T Vector. The connection system is PMD19-T Vector 0.5μL, target gene 4.5μL, SolutionI5.0μL, total volume 10.0μL, mix well and put it in a 16℃ thermostat for overnight connection. The next day, remove E. coli DH5α competent cells from the refrigerator at -80℃, dissolve them in an ice-water mixture, add 5μL of the ligation product, ice bath for 30min, heat shock at 42℃ for 90s, take it out immediately, place on ice for 1~2min, add 900μL The resistant LB liquid medium was placed in a 37°C incubator with shaking culture for 45 minutes, and centrifuged at 4000 rpm for 5 minutes. About 100 μL of supernatant was retained and spread evenly on the Amp resistant (100 μg / mL) LB solid medium plate , Place the plate upside down in a constant temperature incubator at 37℃ for overnight culture, and verify the plasmid construction results by bacterial liquid PCR and double enzyme...
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