甲型H1N1流感病毒实时荧光PCR诊断试剂盒的制备

目的制备甲型H1N1流感病毒实时荧光PCR诊断试剂盒,并进行验证。方法采用磁珠法从甲型H1N1流感疑似患者咽拭子样品中提取病毒RNA,逆转录合成cDNA。根据NCBI最新公布的大流行甲型H1N1流感病毒(2009)基因序列,设计针对编码基质蛋白M基因的引物和探针,检测甲型流感病毒;设计针对血凝素(HA)基因和神经氨酸酶(NA)基因特异性的引物和探针,检测甲型H1N1流感病毒;同时针对人的RNaseP基因设计用于内部控制的引物和探针。所有探针均为Taqman探针,5'端标记FAM,3'端标记BHQ1。对最佳荧光PCR反应条件进行优化,在此基础上组装成甲型H1N1流感病毒实时荧光PCR诊断试剂盒,对其特异性、灵敏度、精密性和稳定性进行验证。与市售试剂盒的检测结果进行对比,并对63份临床甲型H1N1流感疑似患者咽拭子样品进行检测。结果设计的PCR引物及探针能对甲型H1N1流感病毒进行准确检测,与流感病毒的其他型和亚型无交叉反应;试剂盒的灵敏度为0.004个血凝素单位;试验内变异系数小于2.5%,批间变异系数小于5%;试剂盒放置-20℃保存,稳定性良好;检测20份甲型H1N1流感疑似患者咽拭子样品的结果与市售试剂盒一致;检测63份流感疑似患者咽拭子样品,其中大流行甲型H1N1流感病毒阳性36份,普通甲型流感病毒阳性5份。结论所制备的甲型H1N1流感病毒实时荧光PCR诊断试剂盒具有较高的灵敏度、特异性、精密性和稳定性,可用于目前流行的甲型H1N1流感病毒的快速检测。

Preparation of Diagnostic Kit for Influenza A H1N1 Virus by Real-time Fluorescent PCR

Objective To prepare and verify a diagnostic kit for influenza A H1N1 virus by real-time fluorescent PCR.Methods Viral RNA was extracted from the nasopharyngeal swabs of suspected patients with influenza A(H1N1 strain)by magnetic beads-based kit and reversely transcribed to cDNA.The primers and probes specific to the M gene encoding matrix protein were designed according to the gene sequence of influenza A H1N1 virus(2009)published lately in NCBI for detection of influenza A virus,while those specific to HA and NA genes for influenza A H1N1 virus.Meanwhile,the primers and probes specific to human RNase P gene were designed for internal control.All the probes were Taqman probes,of which the 5'-terminus were labeled with FAM and 3'terminus with BHQ1.The reaction condition for real-time fluorescent PCR was optimized,based on which a diagnostic kit for influenza A H1N1 virus by real-time fluorescent PCR was assembled and verified for specificity,sensitivity,precision and stability.The detection results by the prepared kit were compared with those by commercial kit.Sixty-three swabs of suspected patients with influenza A were detected by the prepared kit.Results Influenza A H1N1 virus was accurately detected by the designed PCR primers and probes,and no cross reactions with influenza virus of other types or subtypes were observed.The sensitivity of the prepared kit was 0.004 hemagglutinin units /ml.The intra-and inter-coefficients of variation of detection results by the prepared kit were less than 2.5% and less than 5% respectively.The kit showed high stability at-20℃.The detection results of 20 swabs of suspected patients with influenza A(H1N1)strain by the prepared kit were consistent with those by commercial kit.A total of 63 swabs of suspected patients with influenza A were detected by the prepared kit,of which 36 were positive for pandemic influenza A H1N1 virus and 5 for common influenza A virus.Conclusion The prepared diagnostic kit for influenza A H1N1 virus by real-time fluorescent PCR shows high sensitivity,specificity,precision and stability,which may be used for rapid detection of the current influenza A H1N1 virus.