实时荧光定量PCR共检测人疱疹病毒6型和7型核酸方法的建立和应用

Establishment and application of quantitative real-time PCR for co-detection of HHV-6 and HHV-7

目的:建立同时检测样品中人疱疹病毒6型(human herpesvirus 6,HHV-6)和人疱疹病毒7型(human herpesvirus 7,HHV-7)核酸的单一实时荧光定量PCR(real-time PCR)方法。方法:分析HHV-6、HHV-7型代表株基因组核酸序列,选取同源序列设计单一探针和分别针对HHV-6和HHV-7的扩增引物,通过试验比较和分析,确定使用混合引物对检测没有影响,对扩增温度进行优化后建立Real-time PCR方法。对所建立方法的特异性、检测下限(limit of detection,LOD)、定量下限(limit of quantitation,LOQ)、线性、准确性进行验证,使用该方法对临床样品和生物制品生产用细胞基质进行检测,并与套式PCR的检测结果进行比较。结果:经比较,HHV-6和HHV-7扩增引物混合对检测性能无影响,可分别或同时检测样品中存在的HHV-6/HHV-7核酸。特异性验证中,293细胞及PRV、VSV、VZV、EB1、EB2、EB3、CMV1/2/3、HSVI、HSVII基因组DNA样品对检测无干扰。方法的LOD和LOQ分别为:1×10^(2)copies和1×10^(3)copies,线性范围为1×10^(3)~1×10^(7)copies。使用建立的Real-time PCR方法和套式PCR方法分别对86份临床血浆样品和15种生产用细胞基质样品进行检测,两种方法在临床样品中分别检出8份和4份阳性,χ^(2)分析表明,real-time PCR方法检出率显著高于套式PCR方法,15种细胞基质样品无阳性。结论:所建立的Real-time PCR方法可在单个扩增反应中同时检测HHV-6和HHV-7核酸,检测灵敏度高于传统的套式PCR方法,可用于临床样品和生产用细胞基质样品的检测。

Objective:To establish a single quantitative real-time PCR for the co-detection of human herpesvirus 6(HHV-6) and human herpesvirus 7(HHV-7) nucleic acids in clinical samples and cell matrix samples. Methods The genomic nucleic acid sequences of representative HHV-6 and HHV-7 strains were analyzed. The homogenous sequenceswere selected to design the single probe and individual primers for HHV-6 and HHV-7. Through experimental comparison and analysis,it was determined that the use of mixed primers had no impact on detection. The Real-time PCR was established after the amplification temperature was optimized. The specificity,limit of detection(LOD),Limit of quantitation(LOQ) and linearity(accuracy) of Real-time PCR were validated. The method was used to detect the clinical samples and the cell matrix samples for biological products. The results were compared with those of nested PCR. Results:By comparison,the mixing of HHV-6 and HHV-7 primers had no effect on the detection performance,and HHV-6/HHV-7 could be detected separately or simultaneously. In the specificity test,the genomic DNA of 293 cells showed no interfere,and there were no amplification in samples containing PRV、VSV、VZV、EB1、EB2、EB3、CMV1/2/3、HSVI、HSVII. LOD and LOQ of the method are 1×10^(2) copies and 1×10^(3) copies,respectively. The linear range of the method is 1×10^(3)-1×10^(7) copies. Real-time PCR and nested PCR were used to detect 86 clinical plasma samples and 15 biological production cell matrix samples. 8 samples and 4 samples of clinical samples were detected positive,respectively. And 15 cell matrix samples showed all negative results. Results of χ^(2) analysis showed that the detection sensitivity of Real-time PCR was significantly higher than nested PCR. Conclusion:The established real-time PCR can be used to detect HHV-6 and HHV-7 simultaneously in a single PCR reaction. The sensitivity of Real-time PCR is higher than the nested PCR,and it can be used to detect HHV-6 and HHV-7 in clinical plasma samples and cell matrix samples.