Abstract
Hepatitis E virus (HEV) is the causative pathogen of hepatitis E, and genotypes 3 and 4 strains are detected in Japan. Since highly pathogenic genotype 4 strains are more frequently detected in Hokkaido than in other regions, we developed a multiplex real-time reverse transcription (RT) polymerase chain reaction (PCR) assay to rapidly discriminate between genotypes 3 and 4 (D-PCR). To improve the sensitivity of D-PCR to genotype 4, we compared the performance between QuantiTect Probe RT-PCR Kit (conventional reagent) and Reliance One-Step Multiplex RT-qPCR Supermix (BIO-RAD, A reagent) using HEV RNA-positive samples, the HEV RNA concentrations and genotypes of which were already determined. The linear dynamic range of A reagent for genotype 3 was similar to that of the conventional reagent, whereas that for genotype 4 was 10 times wider than that of the conventional reagent. The PCR efficiencies using the conventional reagent and A reagent were respectively 109.9% vs. 108.3% for genotype 3 and 89.7% vs. 97.1% for genotype 4. The sensitivities of PCR using the conventional reagent and A reagent when using 1,000 μL of plasma were respectively 20 IU/mL vs. 19 IU/mL for genotype 3 and 66 IU/mL vs. 16 IU/mL for genotype 4. As the efficiency and sensitivity of D-PCR have been improved by using A reagent, D-PCR can rapidly determine genotypes at an earlier stage than before, especially for individuals infected with the highly pathogenic genotype 4 strains, and promptly provide genotype information useful for subsequent treatments.
