The Establishment of a SYBR Green Fluorescence Quantitative PCR Detection Method by Targeting the G-protein Coupled Chemokine Receptor Gene of Goat Poxvirus
Based on the genomic sequence of the goat pox virus(GTPV),we have established a fluorescence quantitative PCR method targeting the G-protein-coupled chemokine receptor(GPCR)gene.Six pairs of specific qPCR primers were designed based on the whole genomic se-quence of GTPV.The specificity and sensitivity of these six qPCR primers were screened and de-tected.A pair of qPCR primers with high specificity and sensitivity was finally screened and ob-tained.According to the GPCR gene sequence of the GTPV AV41 vaccine strain,we designed an ordinary PCR primer pair to amplify the GPCR gene to construct the eukaryotic expression vec-tor,which was used to establish the standard curve.Results showed that A pair of specific qPCR primers targeting the GPCR gene was obtained.The eukaryotic expression plasmid named pCAGGS-GPCR was constructed.The standard curve of y=-3.5289x+49.07 was established,whose linear correlation coefficient is R2=0.997 2 and amplification efficiency is 92.7％.The re-sults of the replication experiment suggested that the lowest detective limitation of this primer was 2.0 copies·μL-1,and the coefficient variation of the reproducibility results within and be-tween batches was 2％,indicating the advantages of reasonable specificity,repeatability,and sensitivity.We have successfully established a specific fluorescence quantitative PCR method tar-getting the GPCR gene of GTPV,which provides adequate detecting support for the veterinary clinical diagnosis and the prevention and control of goat poxvirus.
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维普
