Establishment and Application of a Dual-fluorescence Quantitative PCR Method for Detecting Goose Astrovirus Type Ⅰ and Type Ⅱ
In order to establish a rapid and sensitive SYBR GreenⅠ dual-fluorescence quantitative PCR method for goose astrovirus(GAstV)types Ⅰand Ⅱ,specific primers were designed based on the conserved ORF1a sequence of GAstV-1 and ORF2 sequence of GAstV-2.The PCR-amplified fragments were cloned into the pMD18-T vector to construct recombinant plas-mids as positive standard plasmids.The SYBR GreenⅠ dual-fluorescence quantitative PCR method was developed to detect and differentiate GAstV-1 and GAstV-2,and its specificity,sensitivity and repeatability were evaluated,28 suspected-gout gosling clinical samples were also tested using this method.The results showed that the correlation coefficients of standard curves of the established method for GAstV-1 and GAstV-2 were above 0.99,the inter-and intra-assay coefficients of variation were both less than 0.7%,and the lowest detection limit was 10 copies/µL.No specific amplification was observed for duck viral hepatitis virus,avian influenza virus,or goose parvovirus,and there was no cross-reactivity between GAstV-1 and GAstV-2.The positivity rate of the clinical samples detected by the established SYBR GreenⅠ dual-fluorescence quantitative PCR method was 92.8%.Among the positive samples,6 samples showed positive for both GAstV-1 and GAstV-2,18 samples showed positive for GAstV-2 only,and only 2 samples showed positive for GAstV-1 alone.The results indicated that the SYBR GreenⅠ dual-fluorescence quantitative PCR method established in this study could be used for the differentiation of GAstV-1 and GAstV-2,and the domi-nant pathogen causing GAstV infection was still GAstV-2,and mixed infection of GAstV-1 and GAstV-2 also existed.
万方数据
维普
