诺如病毒遗传组I型TaqMan—MGB探针实时荧光RT-PCR的研究

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。

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

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.