Development, evaluation, and standardization of a real-time TaqMan reverse transcription-PCR assay for quantification of hepatitis A virus in clinical and shellfish samples

Abstract

A standardized real-time reverse transcription-PCR (RT-PCR) assay has been developed for an accurate estimation of the number of genome copies of hepatitis A virus (HAV) in clinical and shellfish samples. Real-time procedures were based on the amplification of a fragment of the highly conserved 5' noncoding region and detection through an internal fluorescent probe, including TaqMan and beacon chemistries, in one- and two-step RT-PCR formats. The best performance in terms of sensitivity and reproducibility was achieved by a one-step TaqMan RT-PCR, with a sensitivity enabling the detection of 0.05 infectious unit and 10 copies of a single-stranded RNA (ssRNA) synthetic transcript. Standard reagents, such as a mengovirus strain and an ssRNA transcript, were employed as controls of nucleic acid extraction and RT-PCR, respectively. The test proved to be highly specific after a broad panel of enteric viruses was tested. Sequence alignment of target regions of the primers and probe proved them to be adequate for the quantification of all HAV genotypes. In addition, a quasispecies analysis of the mutant spectrum indicated that these regions are not prone to variability, thus confirming their robustness.

FIG. 1.

Consensus sequence alignment of human HAV genotypes in the amplified region, including the primer and probe target sequences. Nucleotide numbering is according to the HM-175 strain. Terminal 5′ end nucleotides in the available sequences from genotypes II and III are missing.

FIG. 2.

Standard curves for the HAV real-time TaqMan RT-PCR assay. Three molecules were employed: a dsDNA, an ssRNA, and the strain pHM175 43c viral genome. The concentrations of the synthetic molecules were estimated by determining the OD₂₆₀. Virus titer was determined by infectivity, and physical genomes were estimated by applying a factor of ×60 to the infectious titer (7, 13).

FIG. 3.

Proposed standardized procedure for an accurate estimation of HAV genome copies in food or clinical samples. IC, internal control.

FIG. 4.

Time course for HAV genome copies in sera and feces from patients of a shellfish-borne outbreak.