柯萨奇病毒A10荧光定量PCR检测方法的建立

Development of a real-time RT-PCR for detection of coxsackievirus A10

目的建立一种检测柯萨奇病毒A10的荧光定量PCR方法。方法从Gen Bank上下载柯萨奇病毒A10 VP1基因序列并进行同源性分析,选取相对保守的区域设计引物和探针,建立和优化柯萨奇病毒A10荧光定量PCR检测反应体系,并对质粒标准品和临床样本进行检测,评估其特异性和灵敏度。结果优化的引物探针浓度分别为0.5μmol/L和0.2μmol/L,退火/延伸温度为58℃。建立的柯萨奇病毒A10荧光定量PCR检测方法具有良好的特异性,仅质粒标准品和柯萨奇病毒A10阳性临床样本能获得S形扩增曲线,其他临床样本未见任何扩增,而且灵敏度高。probit回归分析估测其检测下限为3.82拷贝/μl(95%置信区间)。结论建立的荧光定量PCR方法能准确检测出柯萨奇病毒A10,可用于临床样本柯萨奇病毒A10的检测。

Objective To develop a real-time RT-PCR for detection of coxsackievirus A10（ CVA10）. Methods VP1 sequence of CVA10 downloaded from Gen Bank was conducted for homologic analyzed. Primers and probes were selected from relatively conservative regions to establish and optimize Coxsackie A10 fluorescent quantitative PCR detection system,and the plasmid standard and clinical samples were tested to evaluate the specificity and sensitivity. Results The optimal concentrations of primers and probes were 0. 5 μmol/L and 0. 2 μmol/L,respectively. The optimal annealing/extension temperature was 58 ℃.The established Coxsackie virus A10 fluorescence quantitative PCR detection method had good specificity,only the plasmid standard and Coxsackie virus A10 positive clinical samples can obtain S-shaped amplification curve,other clinical samples were without any amplification,and the method was sensitive,probit regression analysis estimated that the detection limit was3. 82 copies/μl（ 95% confidence interval）. Conclusion The real-time RT-PCR could accurately identify CVA10. It is suitable for the detection of CVA10 from clinical samples.