中国动物检疫2016，Vol.33Issue(10)：90-94,5.DOI:10.3969/j.issn.1005-944X.2016.10.023
H1和H3亚型猪流感病毒二重TaqMan-MGB探针荧光RT-PCR检测方法的建立
Development of a Duplex TaqMan-MGB Real-time RT-PCR Assay for Detecting H1 and H3 Subtype Swine Inlfuenza Virus
摘要
Abstract
Absract:A Taqman-MGB probe duplex real-time RT-PCR was developed to detect H1 and H3 subtype swine influenza virus(SIV). Two pairs of primers and probes were designed according to the conserved regions of HA gene sequences of H1 and H3 subtype. The assay was specific to detect subtype H1 and H3 SIV,but had no cross-reaction with H9N2 subtype SIV,classical swine fever virus(CSFV),porcine reproductive and respiratory syndrome virus(PRRSV), porcine epidemic diarrhea virus(PEDV)and transmissible gastroenteritis virus(TGEV). The assay has a broad linear detection range from 3.7×101 copies/µL to 3.7×108 copies/µL for RNA standard control of hemagglutinin(HA) of H1 subtype(SIV-H1-RNA)and 3.4×101 copies/µL to 3.4×108 copies/µL for RNA standard contol of HA of H3 subtype(SIV-H3-RNA),respectively. The detection limit of the assay was 37 copies for the SIV-H1-RNA and 34 copies for the SIV-H3-RNA,respectively. The coefficients of variation(CVs)of both inter-assay and intra-assay were less than 2.0 %,showing good reproducibility. 598 nasal swab samples were tested for subtype H1 and H3 detection by the assay. No positive H1 and H3 subtype reaction was found for all 528 samples from imported pigs. 12 and 7 of 78 nasal swab samples of domestic pigs were found to be positive for H1 and H3 respectively. In short,the development of this assay offers a useful method for the simultaneous detection of subtype H1 and H3 SIV in clinical specimens from the pigs.关键词
猪流感病毒/H1亚型/H3亚型/TaqMan探针/荧光RT-PCRKey words
swine influenza virus/subtype H1/subtype H3/TaqMan probe/duplex real-time RT-PCR分类
农业科技引用本文复制引用
王建华,董志珍,赵祥平,陈小金,张俊哲,王玉玲,肖妍,赵丹,谭旭菲,王乃福,陈本龙..H1和H3亚型猪流感病毒二重TaqMan-MGB探针荧光RT-PCR检测方法的建立[J].中国动物检疫,2016,33(10):90-94,5.基金项目
天津市科技支撑项目（13ZCZDNCO1300）；天津市滨海新区惠民项目 （）
