Establishment and Application of Fluorescence Quantitative PCR Detection Method based on Lumpy Skin Disease Virus ORF61 Gene
Based on the genomic sequence of a prevalent Lumpy skin disease virus(LSDV)in Chi-na,we established an MGB probe fluorescence quantitative PCR method for detecting the ORF61 gene.In this study,a specific fluorescent quantitative PCR primer with an MGB probe was de-signed based on the LSDV gene sequence,and the specificity and sensitivity of this primer pair were screened and verified.Finally,we screened a pair of qPCR primers targeting the LSDV ORF61 gene.Using pCAGGS-ORF61,a eukaryotic expression plasmid,the standard curve y=-3.287 5x+47.87 was obtained,the linear correlation coefficient was R2=0.998 6,and the amplification efficiency reached 101％.The results of the specificity test,repeatability test,and sensitivity test showed that this fluorescent quantitative PCR primer pair had the advantages of high specificity,good repeatability,and high sensitivity.The minimum detection limit of this primer pair was 6.71 copies·μL-1,and the coefficient of variation of intra-batch and inter-batch repeatability results was less than 2％.The above results indicate that we have established a fluo-rescence quantitative PCR method for the specific detection of LSDV,providing an effective detection method for preventing and controlling LSD.
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