Comparative evaluation of a novel TaqMan real-time reverse transcription–polymerase chain reaction assay for hepatitis A virus detection

ObjectiveTo develop and evaluate a novel system for detecting and quantifying hepatitis A virus (HAV) nucleic acid.MethodsReal-time TaqMan® reverse transcription–polymerase chain reaction (PCR) procedures were established, based on amplification of the highly conserved 5′-non-coding region. Synthetic single-stranded RNA transcripts synthesized in vitro were used as the quantification standard. Ten-fold dilutions were prepared from HAV strain stock suspension to determine precision, accuracy, sensitivity and specificity. In addition, serum specimens from patients with acute HAV underwent clinical evaluation.ResultsThe novel assay had a detection limit for HAV RNA of 10 TCID50/ml (where TCID50 is median tissue culture infective dose). It was more sensitive and specific than the commercial quantitative PCR kit manufactured by Shanghai Zhijiang Bio-Tech. However, the Artus HAV RT–PCR kit (Qiagen) had the best performance of the three assays and had a detection limit of 5 TCID50/ml. The new HAV real-time PCR d...