Molecular detection of HAV by a new one step real time RT-PCR

Introduction and objectives
Hepatitis A virus (HAV) is a RNA virus with a single-stranded positive sense genome and the only species of the genus Hepatovirus of the Picornaviridae family. Belgium and European countries in general, are countries with a low prevalence and the majority of adults can be infected. HAV is mainly transmitted by the fecal-oral route and even if foodborne outbreaks account for less than 5 % of the reported cases per year, the source of infection cannot be identified in 50 % of the reported cases. Therefore the contribution of foodborne infection is probably underestimated. Viral loads in food samples are lower than in clinical samples and their detection requires refined molecular detection methods.
Methods
A one step real-time RT-PCR to detect HAV, with new primers (HAV F2 and HAV R2) and probe (HAV P2) was performed directly on HAV diluted suspensions and on food samples (dates) and was compared with a ready-to-use commercial kit. Before the one step real time RT-PCR, a preliminary step combining concentration of viral particles with polyethyleneglycol and centrifugation was used on food samples.
Results
Real time RT-PCR one step with HAV F2/R2/P2 is more efficient but less sensitive than the commercial kit. It could be used to confirm a positive sample or to detect HAV in an unknown sample. With cell cultured HAV, the limit of detection (LOD) is 1.25 infectious particles in volume tested by RT-PCR or 102 TCID50/ml. In food samples, LOD is between 25 infectious particles and 250 infectious particles in volume tested by RT-PCR or between 104 and 105 TCID50/ml. Several hypotheses could explain these results: the loss of viral particles during the extraction process, the low efficiency of RNA extraction and interference of food on molecular detection.
Conclusion
Molecular detection of virus in food samples remains a challenge and the protocol of extraction should be improved and adapted at each food category to increase the sensitivity of detection in food matrices characterized by a low viral contamination.