甲型肝炎病毒实时荧光定量PCR检测方法的比较

Comparison of Different TaqMan Real-time PCR Assays for Hepatitis a Virus Detection

目的比较本实验室内部建立的甲型肝炎病毒核酸定量体系和另外两个商品化实时荧光定量PCR检测试剂盒的检测效果。方法本实验室内部建立的HAV核酸检测系统选取HAV基因组的高保守区段5'非编码区作为扩增靶点,定量标准品采用体外转录获得的RNA分子,HAV疫苗株储存液倍比稀释成10-8～10-1后提取RNA作为模板用以比较该反应体系与市售商品化试剂盒的准确性、灵敏度以及特异性。结果本实验室内部建立的HAV核酸检测体系十分适宜于HAV的检测,最低检测极限达到10 TCID50/ml,比国内某厂商生产的HAV定量PCR检测试剂要灵敏,然而Qiagen公司的RealArt HAV LC RT-PCR检测试剂效果最好,其检测极限可以达到5 TCID50/ml。结论本实验室内部建立的HAV核酸定量体系可重复性好,敏感度高,特异性强,在临床样本,环境样本以及食品样本的检测方面具有广阔的应用前景。

Objective To compare our in - house detection and quantification system of hepatitis A virus （HAV） with another two commercial quantitative real - time PCR kits. Methods The in - house HAV detection method developed in our laboratory was based on the amplification of the highly conserved 5＇ - noncoding region. Single - stranded RNA transcripts synthesized in vitro were used as quanti- fication standard. Dilutions of an HAV vaccine strain ranging from 10 -s to 10 -1 were prepared to determine the precision, accuracy, sen- sitivity, and specificity of this assay. Results Our in - house TaqMan based real - time PCR assay was suitable for measurement of HAV RNA and had a detection limit of 10 TCID50/ml. This assay was more sensitive and specific than one of the domestic commercial quantita- tive PCR kit. However, another assay, the Artus HAV RT -PCR kit from Qiagen （Germany） , had the best performance among these 3 assays and its detection limit achieved 5 TCID50/ml. Conclusion Considering the high reproducibility, sensitivity, and specificity, our novel amplification system could be widely applied to detect HAV in clinical, environmental, and food samples.