Animal insect-borne disease multi-RT-PCR distinguishing and detecting reagent as well as preparation method and application
A technology of RT-PCR and identification detection, which is applied in the direction of biochemical equipment and methods, methods based on microorganisms, measurement/inspection of microorganisms, etc. It can solve the problems of poor specificity, low sensitivity, time-consuming and labor-intensive operation, etc., and achieve the application of samples wide range of effects
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[0057] 1. Design primers
[0058] The present invention selects the conserved fragments of the BTVVP7 gene, EHDVVP7 gene, VSVN gene and AKVN gene as the target, and selects the BTVVP7 gene respectively by performing homology analysis and comparison on the cDNA sequences of the BTV, EHDV, VSV and AKV genes reported in GenBank , EHDVVP7 gene, VSVN gene and AKVN gene conserved fragments, primer Express software and primer prere5.0 software were used to design primers, primers were synthesized by β-acetonitrile phosphorous acid amine chemical synthesis method, and OligoDNA primers were carried out by automatic DNA synthesizer Synthesis. After carrying out the optimal pairing screening experiment and cross-reaction test on the multiple pairs of primers designed and synthesized, the four pairs of primers with the best amplification efficiency and specificity for each of the four viruses were determined, and the lengths of the amplified target fragments were 351bp, 536bp, 300bp and ...
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