Comparative evaluation of new TaqMan real-time assays for the detection of hepatitis A virus.
Three novel real-time TaqMan RT-PCR assays targeting the 5'-UTR, the viral protease and the viral polymerase regions of the hepatitis A virus (HAV) were developed, evaluated and compared against a new published 5'-UTR TaqMan assay (JN) and a widely used conventional RT-PCR assay (HAVc). All conventional RT-PCR (HAV, SH-Prot and SH-Poly systems) and TaqMan (SH-Prot, SH-Poly, JN and SH-5U systems) assays evaluated were consistent for the detection of the three different HAV strains (HM-175, HAS-15 and LSH/S) used and reproducible for both RNA duplicates with the exception of two reproducibility discrepancies observed with both 5'-UTR real-time systems (JN and SH-5U). Limits of detection for conventional HAV, SH-Prot and SH-Poly RT-PCR systems were found to be equivalent when tested with serially diluted suspensions of the HM-175 strain. Although the four real-time RT-PCR TaqMan assays evaluated herein produced similar and consistent quantification data down to the level of one genomic equivalent copy with their respectively cloned amplicons, significant and important differences were observed for the detection of HAV genomic RNA. Results showed that the new real-time TaqMan SH-Poly and SH-Prot primer and probe systems were more consistent and sensitive by 5 logs as compared to both 5'-UTR designs (JN and SH-5U) used for the detection of HAV genomic RNA as well as for the detection in cell culture by cytopathic effect. Considering their higher analytical sensitivity, the proposed SH-Poly and SH-Prot amplification systems could therefore represent valuable tools for the detection of HAV in clinical, environmental and food samples.
Houde
Alain
A
Agriculture and Agri-Food Canada, Food Research and Development Centre, 3600 Casavant Blvd. West, Saint-Hyacinthe, Quebec J2S 8E3, Canada. houdea@agr.gc.ca
Guévremont
Evelyne
E
Poitras
Elyse
E
Leblanc
Danielle
D
Ward
Pierre
P
Simard
Carole
C
Trottier
Yvon-Louis
YL
D001483:Base Sequence; D001681:Biological Assay; D017931:DNA Primers; D015342:DNA Probes; D005069:Evaluation Studies as Topic; D017957:Hepatitis A Virus, Human / Q000235:genetics* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques; D012367:RNA, Viral / Q000032:analysis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012689:Sequence Homology, Nucleic Acid; D019914:Taq Polymerase; D013997:Time Factors
D017931:DNA Primers; D015342:DNA Probes; D012367:RNA, Viral; D019914:Taq Polymerase