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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 establish real-time SYBR Green quantitative PCR method for the detection of EV71 subtypes. Methods According to the pathogen of hand, foot and mouth disease in mainland China, a subtype of EV71VP1 gene specific primer was designed. The VP1 gene fragment was amplified by PCR and ligated with pEASY-T1 vector to construct pEASY-T1-VP1 recombinant plasmid. Fluorescent quantitative PCR was used to draw the standard curve to evaluate the sensitivity, specificity and stability of the method. The method was used to detect the throat swab samples of patients with hand-foot-mouth disease in outpatients. Results The established real-time fluorescence quantitative PCR standard curve had good linearity (R2 = 0.993) and amplification efficiency of 107.1%. The detection limit of the method was between (8.48 × 100) ~ (8.48 × 101) copies / μl and the coefficient of variation 1%. The non-HFMD pathogen had no amplification signal. The positive rate of throat swab in HFMD patients was 52.3% (45/86). Positive samples were compared with NCBI after sequencing, the coincidence rate was 100%. Conclusion The established SYBR Green real-time PCR is sensitive and specific and can be used to detect multi-subtype EV71 infection.