实时荧光定量RT—PCR快速检测H7N9禽流感病毒

Rapid assay for H7N9 avian influenza virus genes by real-time fluorescence quantitative RT-PCR

目的构建一种灵敏、特异、快速的实时荧光定量RT—PCR反应体系用于临床H7N9禽流感疑似病例早期诊断及外环境H7N9病毒的监测。方法以H7N9禽流感病毒H7基因和N9基因为靶基因设计引物以及TaqMan探针，并对引物、探针及反应条件进行优化，验证该反应体系检测的特异性、灵敏度、重复性和检测速度，然后与两种商品化试剂盒（试剂盒I和试剂盒Ⅱ）进行各项指标及184份现场样本作平行比对。结果新建立的H7N9禽流感病毒实时荧光定量RT-PCR反应体系呈典型的扩增曲线、扩增时间约50min，H7基因与N9基因的最低检出限分别为28拷贝／μL与41拷贝／μL。重复性测定显示，H7和N9检测Ct值的变异系数（CV）分别为0．17％～0．89％和0．33％～0．59％，扩增效率分别为0．9491和0．9713；与其他常见禽流感病毒或肠道病毒无明显交叉反应。184份现场可疑样本阳性检出率为5．43％（10／184），与试剂盒Ⅱ结果一致。结论本研究建立的实时荧光定量RT—PCR反应体系具有检测速度快、灵敏度高、特异性强、重复性好、结果可靠等特点，可用于临床疑似病例的应急检测、早期诊断及养鸡场、活禽交易市场等外环境H7N9禽流感病毒监测。

Objective To develop a sensitive, specific and rapid reaction system of real-time fluorescence quantitative RT-PCR for early diagnosing clinical HTN9 avian influenza suspected patients and monitoring environmental pollutions. Methods Primers and TaqMan probes were designed according to the H7 and N9 genes of H7N9 avian influenza virus, then the primers, probes and reaction conditions were optimized, the specificity, sensitivity, repeatability and detection speed were verified. The RT-PCR method developed was compared with two commercial kits （Kit I and Kit II）in every parameter and 184 field samples. Results The real-time fluorescence quantitative RT-PCR developed for detecting H7N9 avian influenza virus had typical amplification curves, the detection time was 50 minutes, the detection limit of H7 gene and N9 gene was 28 copies/p.L and 41 copies/p^L, respectively. Repeatability assay showed that coefficients of variation of Ct value were 0.17%-0.89%and 0.33%- 0.59%, respectively, amplification efflciencies were 0.9491 and 0.9713. No significant cross-reaction was found between H7N9 avian influenza virus and other common avian influenza viruses or enteroviruses. The positive rate was 5.43%（10/184） in 184 field suspicious samples, which was consistent with Kit II. Conclusions The real-time fluorescence quantitative RT-PCR reaction system develops for rapid assay of H7N9 avian influnenza virus gene, good sensitivity, specificity, repeatability and reliable results, and can be used for emergency detection and early diagnosis of clinical suspected patients with H7N9 avian influenza virus infection, and for surveillance of the virus in external environments like chicken farms and live poultry markets.