脊髓灰质炎病毒实时荧光定量RT-PCR检测方法的建立及应用

摘要:目的　在省级脊髓灰质炎（脊灰）实验室建立一种快速、灵敏、准确的脊灰病毒（Poliovirus，PV）型内鉴定（Intratypic Differentiation，ITD）及基因型鉴定的方法。方法　以PV衣壳蛋白（Capsid Protein）VP1编码区基因序列为目标，设计并合成引物和 Taqman 探针，建立实时荧光定量RT-PCR（Real-time RT-PCR，rRT-PCR）检测体系，并考察该方法的重复性、灵敏性和特异性。结果　该方法能快速、灵敏地鉴定出PV血清型及毒株类型，在1.0×108 copies/μl～1.0×103 copies/μl检测范围之间有良好的线性关系，相关系数为0.993，最低检测限为103.5CCID50/0.1 ml。结论　成功在省级实验室建立了PV的r RT-PCR检测技术，该技术特异性强，敏感性高，操作简便快速，适用于PV的型内鉴定和基因型鉴定，可应用于实验室诊断，为免疫策略快速提供依据。

Establishment and application of poliovirus Real-time fluorescence quantitative RT-PCR detection method

Abstract: Objective To establish a rapid, sensitive and accurate identification for poliovirus (PV) in provincial which can Intratypic Differentiation (ITD) and identify genotype. Methods We designed and synthesized of primers and Taqman probe by usingVP1 coding region of PV capsid protein as the target and establish a Real-time RT-PCR detection system. At the same time, we studied the sensitivity, specificity and reproducibility of the method. Results Real-time RT-PCR can identify the serotype and genotype of PV sensitively and quickly, and there was a good linear relationship between 1.0×108 copies/μl～1.0×103 copies/μl detection range. The correlation coefficient is 0.993, and the minimum detection is 103.5CCID50/0.1ml. Conclusion We established Real-time RT-PCR PV detect technology successfully in provincial laboratory, and that technology was with strong specificity, high sensitivity and simple, fast operation, so it was suitable for Intratypic Differentiation (ITD) and identify genotype of PV and it could be used in laboratory diagnosis and provided the basis for immunization strategies.