OBJECTIVE: To develop and evaluate a novel system for detecting and quantifying hepatitis A virus (HAV) nucleic acid. METHODS: Real-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. RESULTS: The 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 detection system was also successfully applied in 90 serum specimens from patients with confirmed acute HAV infection. CONCLUSION: Considering its high reproducibility, sensitivity, specificity and simplicity, this novel amplification system may be suitable for wide clinical application as a diagnostic tool, and for the surveillance and investigation of infectious diseases in developing countries where HAV is endemic.
OBJECTIVE: To develop and evaluate a novel system for detecting and quantifying hepatitis A virus (HAV) nucleic acid. METHODS: Real-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. RESULTS: The 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 detection system was also successfully applied in 90 serum specimens from patients with confirmed acute HAV infection. CONCLUSION: Considering its high reproducibility, sensitivity, specificity and simplicity, this novel amplification system may be suitable for wide clinical application as a diagnostic tool, and for the surveillance and investigation of infectious diseases in developing countries where HAV is endemic.