诺如病毒遗传组I型TaqMan-MGB探针实时荧光RT-PCR的研究  

The Study for Genogroup I of Norovirus Detection Using TaqMan-MGB Probe- Based Real-Time Fluorescent Reverse Translation Polymerase Chain Reaction

作　　者：谭翰清[1] 郭赐贶 谭海芳[1] 陈华 程洁萍 林凤[1] 孔秀珍[1] 丁丽娜[1] 

机构地区：[1]广东省肇庆市疾病预防控制中心,肇庆526020

出　　处：《热带医学杂志》2008年第10期1030-1033,1040,共5页Journal of Tropical Medicine

基　　金：广东省肇庆市科技创新计划项目(No.2006E1818)

摘　　要：目的建立诺如病毒遗传组I型TaqMan-MGB探针实时荧光RT-PCR快速、特异、灵敏的检测方法,为疾病预防控制提供可靠的依据。方法根据GenBank诺如病毒遗传组Ⅰ型代表株保守序列设计特异引物对和TaqMan-MGB探针,建立一步法实时荧光RT-PCR快速检测反应体系,优化反应条件,评价反应体系的灵敏度、特异性、重复性,并与常规RT-PCR比较。结果诺如病毒遗传组Ⅰ型TaqMan-MGB探针实时荧光RT-PCR检测时限短,仅1h就出结果,与轮状病毒、腺病毒、星状病毒、甲肝病毒、诺如病毒遗传组Ⅱ型无交叉反应,最低检出下限为100拷贝/反应,比常规RT-PCR灵敏100倍,5份浓度不同的诺如病毒遗传组Ⅰ型标本重复检测5次,平均Ct值变异系数范围为0.39%～1.02%。结论诺如病毒遗传组Ⅰ型TaqMan-MGB探针实时荧光RT-PCR快速、特异、灵敏、重复性好,可应用于突发公共卫生应急检测和诺如病毒遗传组Ⅰ型监测,提高快速检测能力。Objective To establish a rapid, specific and sensitive molecular method for the detection of genogruop I of norovirus using TaqMan-MGB probe-based real-time fluorescent polymerase chain reaction, and to provide credible molecular evidence for disease control and prevention. Method According to the genogroup Ⅰ conserved sequence of norovirus, specific primers and TaqMan-MGB probe were designed, and were optimized to establish one step real-time RT-PCR system. The specificity, sensitivity and repeatability of this system were evaluated and compared with the conventional RT-PCR. Result The one step TaqMan-MGB probe-based real-time RT-PCR for genogruop I of norovirus was so rapid that it can be reported in just an hour, and the sensitivity was 100 copies per reaction, and there was no non-specific cross reaction with other viruses, including rotavirus, adenovirus, astrovirus, hepatitis A virus and the genogruop H of norovirus. Five samples containing different concentration of norovirus were tested for 5 times, and the coefficient of variability for the average of Ct values were 0.39%-1.02%. Conclusion The rapid, specific, sensitive and repeatable one step TaqMan-MGB probe-based real-time RT-PCR for the detecting genogruop I of norovirus was successfully established, and could be applied to improve the capacity of rapid detection for the emergency process to the abrupt public hygiene affairs or regular surveillance.

关 键 词：人类杯状病毒 诺如病毒 实时荧光PCR 逆转录PCR 

分 类 号：R373.25[医药卫生—病原生物学]