Broad bean latent virus M double-antibody sandwich enzyme-linked immunoassay kit and preparation and detection methods thereof
A latent virus and immune detection technology is applied in the field of biotechnology products for plant virus detection, which can solve the problems of no VCV-M specific antiserum and undetectability, and achieve large-scale commercial promotion, simple operation, and good performance. The effect of applying the foreground
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[0047] The present invention will be further described below in combination with specific embodiments and accompanying drawings.
[0048] (1) Prokaryotic expression and purification of VCV-M coat protein
[0049] ①Total RNA extraction (Trizol method)
[0050] ②Primer design
[0051] According to the obtained VCV-M isolate sequence, design and synthesize specific primers in amplifying the VCV-M coat protein nucleotide sequence: VCV-M / CP / F (SEQ ID No.1) and VCV-M / CP / R (SEQ ID No.2); restriction sites NcoI and NotI were respectively introduced to connect the prokaryotic expression recombinant vector pET-28a. The primers were purified by PAGE and sent to Nanjing Qingke Biotechnology Co., Ltd. for synthesis.
[0052] ③RT-PCR
[0053] The total RNA of faba bean leaves was used as a template, and VCVMCPR primer was used to synthesize cDNA by reverse transcriptase M-MLV. The reverse transcription system is shown in Table 1.
[0054] Table 2 Reverse transcription system
[0055]...
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