Optimization Of A Reverse Tráncriptase Pcr Method For Detecting Hepatitis E Virus In Domesticated Wild Boar Samples

Abstract

Introduction: Hepatitis E virus (HEV) poses a significant public health concern, with genotypes 1 and 2 causing acute and chronic hepatitis in humans, and genotypes 3 and 4 exhibiting zoonotic potential through undercooked meat consumption and animal contact. This thesis aimed to optimize a PCR protocol for sensitive and specific detection of HEV RNA in fecal samples from domesticated wild boars, a potential reservoir for zoonotic HEV strains. Method: RNA was extracted and cDNA was synthesized from 27 domesticated wild boar fecal samples collected in southern Vietnam. An established protocol was optimized for annealing temperature, primer concentration, and magnesium chloride. The optimized protocol was then evaluated for specificity, sensitivity, and performance on real fecal samples. Result: Optimization involved increasing the annealing temperature to 57.4°C and adjusting primer and MgCl₂ concentrations. Optimization yielded high specificity, sensitive detection (approximately 11.8ng/µL), and a promising 48.2% detection rate in wild boar samples (13 out of 27 samples), suggesting potential high HEV prevalence. Conclusion: A protocol for HEV detection in wild boars was optimized, achieving high specificity, sensitive detection, and a promising 48.2% detection rate in initial validation. However, confirmation of these positive results using established nested PCR and more accurate DNA quantification methods is necessary for definitive validation. This work provides a valuable tool for future studies investigating HEV prevalence in domesticated wild boars and their potential role in HEV epidemiology.