Improved TaqMan real-time assays for detecting hepatitis A virus

Abstract

Rapid advancement in genomics and bioinformatics in recent years holds great promise for research and development in many disciplines including public health. For the detection of pathogens, methods based on nucleic acid amplification need to be re-evaluated periodically to ensure the validity of signature primers and probes as more and more outbreak strains are sequenced and collected into databases in public domains. In this study, a previous assay designed computationally for detecting hepatitis A virus (HAV) was re-examined. Alignment of 57 complete or near complete HAV genomes allowed identification of conserved sequences for developing new primers and TaqMan probes. Two sets of real-time reverse transcription PCR reagents were developed by targeting highly conserved regions with primers and probes having optimal melting temperatures and minimum secondary structures. These two assays had 10 to 1000 fold lower detection limits than the previous assay when tested using representative human HAV genotypes IA, IB, and IIIA. The better of the two improved assays had a detection limit of 3.7 × 10−2 to 6.6 × 10−2 TCID50 or less. The improved detection sensitivity was likely due to improvement in the following four areas: 1) The Gardner1 probe has a single nucleotide mismatch at the 5′ end in all 19 strains of genotypes IIIA and IIIB. 2) For the Gardner1 forward primer, there is a mismatch corresponding to the 3′ end of the oligonucleotides in two strains belonging to genotype IA. 3) The Gardner1 probe had a melting temperature of 66.2 °C, which is less than the optimum of 68–70 °C (Dorak, 2006). 4) The Gardner1 forward and reverse primers had high potential of forming primer dimers.The improved HAV detection assays developed in this study would support better food safety surveillance initiatives and response to disease outbreaks of viral food-borne illness.