Fluorescence quantitative RT-PCR detection reagent for West Nile virus and preparation method and use thereof
A West Nile virus and RT-PCR technology, which is used in the preparation of reagents and the field of reagents for West Nile virus detection, achieves the effect of wide application range, high throughput and strong sensitivity of samples
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[0028] 1. Construction of WNV E gene recombinant plasmid
[0029] According to "Molecular Cloning Experiment Guide" (Third Edition) translated by Huang Peitang et al., China Science Press, January 2003, pages 1217 to 1259, the West Nile virus E gene was cloned and sequenced, and the WNVE gene recombinant plasmid was constructed. , and the recombinant plasmid was named pBAD-E.
[0030] 2. Design primers and probes
[0031]The present invention selects the conserved segment of the WNV E gene as the target, and compares the homology analysis and comparison between the DNA sequences of the virus genes of the WNV epidemic strains reported in GenBank and the DNA sequences of the West Nile virus WNV genes cloned and sequenced in 1 above. , select the conserved fragment (71bp) of WNV gene, apply primerExpress software and primer prere5.0 software, design primers and probes, the synthesis of primers and probes adopts β-acetonitrile phosphite chemical synthesis method, and uses automat...
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