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

摘要

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.%根据H1和H3亚型猪流感病毒（SIV）血凝素（HA）编码基因的保守序列，分别设计合成特异性引物和TaqMan-MGB探针，建立了一种检测H1和H3亚型SIV的二重荧光RT-PCR方法。结果显示，该方法仅对H1和H3亚型SIV呈特异性扩增，对H9亚型SIV、猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（SFV）、猪流行性腹泻病毒（PEDV）和猪传染性胃肠炎病毒（TGEV）无交叉扩增反应；用该方法检测H1和H3亚型SIV的HA基因的 RNA标准对照（SIV-H1-RNA，SIV-H3-RNA），最适线性检测范围分别为3.7×101~3.7×108拷贝数/反应和3.4×100~3.4×108拷贝数/反应，最低检出限分别为38和34个拷贝数；该方法的组内试验和组间试验的变异系数均小于2.0%，显示其具有良好的可重复性。用该方法对520份进口猪的鼻拭子样本和78份国内猪鼻拭子样本进行SIV检测发现，进口猪鼻拭子样本的SIV检测结果均为阴性，12份国内猪鼻拭子样本的H1亚型SIV检测结果为阳性，5份国内猪鼻拭子样本的H3亚型SIV检测结果为阳性。本方法的建立为H1和H3亚型SIV检测提供了一种快速、敏感和特异的技术手段。