title: Comparative evaluation of a novel TaqMan real-time reverse transcription-polymerase chain reaction assay for hepatitis A virus detection.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Institute for Viral Disease Control and Prevention, Chinese Centre for Disease Control and Prevention, Beijing, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Qiu
authors.forename: Huihui
authors.initials: H
authors.lastname: Zheng
authors.forename: Yao
authors.initials: Y
authors.lastname: Yi
authors.forename: Zhiyuan
authors.initials: Z
authors.lastname: Jia
authors.forename: Jingyuan
authors.initials: J
authors.lastname: Cao
authors.forename: Shengli
authors.initials: S
authors.lastname: Bi
mesh_terms: D006506:Hepatitis A / Q000821:virology; D030041:Hepatitis A virus / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D012313:RNA / Q000378:metabolism; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012015:Reference Standards; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019914:Taq Polymerase / Q000378:metabolism*
chemical_list: C034477:RNA primers; D012367:RNA, Viral; D012313:RNA; D019914:Taq Polymerase
keywords: Hepatitis A virus; quantification; real-time reverse transcription–polymerase chain reaction; sensitivity; specificity