基于cDNA-AFLP检测不明RNA病毒方法学研究

Establishment of methodology for detection of unknown RNA virus based on cDNA-AFLP

目的建立cDNA扩增限制片段长度多态性技术(cDNA-AFLP)方法,提高少发或新发病病原体检测灵敏度,为应对可能发生的突发公共卫生事件提供技术储备。方法参考相关文献,分3部分建立cDNA-AFLP方法,包括:标本前处理;两步RT-PCR制备cDNA;cDNA多态长度片段扩增。以Real-time PCR方法为金标准,评价cDNA-AFLP检测乙型流感病毒和EV71的灵敏度与特异度。结果 7件Real-time PCR检测乙型流感病毒阳性标本,2件经cDNA-AFLP检测阳性,灵敏度为28.57%,特异度为100.00%。7件Real-time PCR检测EV71阳性标本,3件经cDNA-AFLP检测阳性,cDNA-AFLP灵敏度为42.86%,特异度为100.00%。结论 cDNA-AFLP能有效检测不明RNA病毒,其灵敏度受标本内病毒载量影响,对CT值小于25的标本检出率较高。该方法不需要特异引物,不需要新投入大型仪器设备,覆盖病原体类别广,成本低,应该成为常规传染病监测项目的必要补充。

Objective To establish the cDNA amplification restriction fragment length polymorphism (cDNA-AFLP) method to improve the sensitivity for detecting unknown RNA virus and to provide technical reserves for responding to possible public health emergency. Methods Reference to relevant literature, the cDNA-AFLP method was established in three parts, including: sample preparation;preparation of cDNA by two-step RT-PCR;and amplification of cDNA polymorphic length fragments. The sensitivity and specificity of cDNA-AFLP for the detection of influenza B virus and EV71 were evaluated by using the real-time PCR method as the golden standard. Results Among seven positive samples tested for influenza B by real-time PCR, 2 samples were positive by cDNA-AFLP. The sensitivity and specificity of cDNA-AFLP were 28.57% and 100%, respectively. Among seven positive samples tested for EV71 by real-time PCR, 3 samples were positive by cDNA-AFLP. The sensitivity and specificity were 42.86% and 100%, respectively. Conclusions cDNA-AFLP can be used for detecting unknown RNA viruses. The sensitivity of the method is affected by the viral load in the samples. The detection rate of samples with CT value less than 25 is high. The cDNA-AFLP method does not require specific primers or any large-scale instrument and equipment. It covers a wide range of pathogens and has a low cost, which should be a necessary supplement to conventional infectious disease surveillance networks.