应用实时荧光定量PCR检测多亚型EV71的研究

Development of a quantitative fluorescence-based PCR assay to detect multiple Enterovirus 71 subtypes

目的建立SYBR Green实时荧光定量PCR方法,以期用于EV71亚型检测。方法根据中国大陆手足口病病原,设计多亚型EV71VP1基因特异性引物,PCR扩增VP1基因片段,并与pEASY-T1载体连接,构建pEASY-T1-VP1重组质粒,以此为标准品进行实时荧光定量PCR,绘制标准曲线,评价方法的敏感性、特异性和稳定性,并利用该方法检测门诊初诊手足口病患者的咽喉拭子标本。结果建立的实时荧光定量PCR标准曲线线性关系良好(R2=0.993),扩增效率为107.1%;方法的检测下限为(8.48×100)^(8.48×101)拷贝/μl之间,变异系数<1%;非手足口病病原体无扩增信号,手足口病患者咽喉拭子阳性率为52.3%(45/86)。阳性样品测序后与NCBI比对,符合率为100%。结论建立的SYBR Green实时荧光定量PCR敏感、特异,可用于多亚型EV71感染的检测。

Objective To develop a SYBR Green fluorescence-based real-time quantitative PCR assay to detect multiple Enterovirus 71（EV71）subtypes. Methods Based on epidemiological data on hand foot mouth disease in China's Mainland,primers specific to the type-common region of the VP1 gene of EV71 were designed and amplified with PCR.Fragments were then ligated into a pEASY-T1 vector to construct the recombinant plasmid pEASY-T1-VP.A standard curve was generated to quantify the number of copies of the gene.The sensitivity,stability,and specificity of the assay were tested,and the assay was used to test throat swabs from patients who were initially diagnosed with hand,foot,and mouth disease by outpatient services. Results A good linear correlation was observed in the standard curve for the quantitative fluorescence-based PCR assay,R-squared was 0.993,and the efficiency of amplification was 107.1%.The assay had a detection limit between 8.48×10^0 copies/μl and 8.48×10^1 copies/μl;its coefficient of variation was less than1%in repeated assays;the quantitative fluorescence-based PCR assay was highly specific for EV71 without cross-reactions to pathogens unrelated to hand,foot,and mouth disease.EV71 was detected in 86 genital tract swabs at a rate of52.3%.Positive samples were sequenced and compared to NCBI,revealing a rate of concordance of 100%. Conclusion Results indicated that a SYBR Green fluorescence-based real-time quantitative PCR assay is highly specific and highly sensitive.This assay could be used to detect and quantitatively analyze EV71 subtypes.