Nested PCR optimization for detecting hepatitis E virus

Abstract

The occurrence of Hepatitis E virus (HEV) infections is on the rise in developing countries, frequently linked to the consumption of undercooked meats and exposure to animal feces. Typically, HEV diagnosis relies on the immunodetection of anti-HEV antibodies and reverse transcriptase polymerase chain reaction. In this study, nested polymerase chain reaction (Nested PCR) for detecting HEV in domesticated pig and farmed wild boar samples was optimized to provide an alternative method for reliable and precise detection of HEV, particularly in animal samples. All samples were collected in Ho Chi Minh City, Vietnam. Total RNA was extracted from liver tissues of domestic pigs (n=48), rectal swabs samples (n=60), and feces samples (n=25) of farmed wild boars. Complementary DNA (cDNA) was synthesized using random hexamers. Nested PCR was performed under four different conditions: protocol 1–4, with variations in reaction components and concentrations. Two PCR programs, designated as A and B, were examined, featuring distinct cycling times and annealing temperatures for the outer and inner amplification stages. The resulting amplification products (306 bp) were visualized through gel electrophoresis. Protocol 1 and 2, when employed in conjunction with both program A and B, failed to identify any positive samples, including the positive control (0%). In contrast, protocol 3, in combination with program A, and protocol 4, paired with program B, successfully identified 28 positive results out of 133 tested samples (21%). It is noteworthy that protocol 4, when used with program B, yielded clearer and more specific bands. The study successfully optimized a nested-PCR protocol for detecting HEV in animal samples, comprising tissues, rectal swabs, and fecal samples.