芯片式数字PCR测定柯萨奇病毒A组6型病毒载量方法的建立及应用  

Establishment and application of a chip-based digital PCR method for quantifying coxsackievirus A6 viral load

作　　者：刘少华 刘宇 刘善茹 张中洋 LIU Shaohua;LIU Yu;LIU Shanru;ZHANG Zhongyang(National Vaccine&Serum Institute,Beijing 100176,China)

机构地区：[1]国药中生生物技术研究院有限公司,北京100176

出　　处：《中国病毒病杂志》2024年第6期578-584,共7页Chinese Journal of Viral Diseases

摘　　要：目的建立芯片式数字PCR(chip-based digital PCR,cdPCR)方法检测柯萨奇病毒A组6型(coxsackievirus A6,CVA6)病毒载量。方法通过优化反应条件确定cdPCR的最佳探针浓度和退火温度,利用CVA6病毒VP1基因的RNA标准品确立cdPCR的线性范围和检出限。随后对cdPCR测定CVA6病毒载量进行方法学验证。利用cdPCR和qPCR方法检测小鼠组织中CVA6病毒载量,对检测结果进行一致性分析。结果确立了探针的最佳浓度为0.125μmol/L,最佳退火温度为53℃;检测范围为3.05~6726拷贝/μl,检出限为1.59拷贝/μl。验证结果提示cdPCR方法的精密度、准确度和特异性均良好。一致性分析结果显示2种方法的一致性良好(r=0.93,P<0.001)。结论基于cdPCR的定量方法能准确反映RNA中CVA6病毒的载量,可应用于疫苗的保护效果评价。Objective To establish a chip-based digital PCR(cdPCR)method to detect the viral load of coxsackievirus A6(CVA6).Methods Optimal reaction conditions for cdPCR were determined by optimizing probe concentration and annealing temperature.The linear range and detection limit of cdPCR were established by using RNA standards of the CVA6 virus VP1 gene.Subsequently,methodological validation of cdPCR for quantifying CVA6 virus load was conducted.Additionally,virus load measurements in mouse tissues by both cdPCR and qPCR were performed,followed by consistency analysis of the results.Results By optimizing reaction conditions,the optimal probe concentration was determined to be 0.125μmol/L and the optimal annealing temperature was 53℃.Using an RNA standard of the CVA6 virus VP1 gene,the detection range of cdPCR was established as 3.05 to 6726 copies/μl,with a detection limit of 1.59 copies/μl.Validation results indicated that the cdPCR method exhibited good precision,accuracy,and specificity.Consistency analysis demonstrated a strong correlation between the two methods(r=0.93,P<0.001).Conclusion The quantitative method based on cdPCR can accurately reflect the load of the CVA6 virus in RNA and can be used to evaluate the protective efficacy of vaccines.

关 键 词：柯萨奇病毒A组6型 芯片式数字PCR 绝对定量检测方法 

分 类 号：R373.2[医药卫生—病原生物学]