Sugarcane streak mosaic virus double-antibody sandwich enzyme-linked immunosorbent assay kit as well as preparation method and detection method of kit
A mosaic virus and immune detection technology, applied in the field of biotechnology products for plant virus detection, can solve problems such as inability to detect, and achieve the effects of simple operation, high sensitivity, and large-scale commercial promotion.
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[0047] The present invention will be further described below in conjunction with specific embodiments and drawings.
[0048] (1) Prokaryotic expression and purification of SCSMV coat protein
[0049] ① Total RNA extraction (polysaccharide polyphenol plant tissue lysis method)
[0050] ②Primer design
[0051] According to the obtained SCSMV-M114 isolate sequence, design and synthesize specific primers for amplifying the nucleotide sequence of the SCSMV coat protein: SCSMCPF F (SEQ ID No. 1) and SCSMCPR F (SEQ ID No. 2); respectively introduced Restriction site Nco I and Sac I are used to connect the prokaryotic expression recombinant vector pET-28a. The primers were purified by PAGE and sent to Shanghai Shenggong Bioengineering Co., Ltd. for synthesis.
[0052] ③RT-PCR
[0053] Using the total RNA of sugarcane leaves as a template, using SCSMCPR primers, and using reverse transcriptase M-MLV to synthesize cDNA, the reverse transcription system is shown in Table 1.
[0054] Table 2 Revers...
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