Nested real-time PCR for hepatitis A detection

Abstract

The hepatitis A virus (HAV) is one of the most important human foodborne pathogens causing a number of worldwide outbreaks each year. The detection of HAV in food samples remains a complex issue, because commonly used detection tools, such as conventional or even real-time PCR assays, are often unable to detect HAV with sufficient sensitivity. The aims of this study were to develop highly sensitive and specific nested real-time PCR (NRT-PCR)-based method for HAV detection in food and to compare it with currently available methods. By combining conventional PCR, nested PCR and real-time PCR techniques, we have developed a specific NRT-PCR assay for the detection of HAV. The procedure involves two consecutive PCRs, the first of which is performed as a conventional RT-PCR using primers specific for HAV 5' noncoding region. The second reaction involves a real-time PCR using a nested primer pair specific for the first PCR product and a TaqMan probe. We have developed a novel NRT-PCR method capable of detecting as little as 0.2 PFU of HAV, which is significantly more sensitive than any other PCR technique tested in our system. NRT-PCR provides a potentially useful method for detecting HAV at extremely low levels, as frequently found in food samples, and can be potentially adopted as a regulatory method to ensure food safety.