Combined Immunomagnetic Separation–Molecular Beacon–Reverse Transcription-PCR Assay for Detection of Hepatitis A Virus from Environmental Samples

In this study, a molecular-beacon-based real-time reverse transcription (RT)-PCR assay was developed to detect the presence of hepatitis A virus (HAV) in environmental samples. A 125-bp, highly conserved 5 noncoding region of HAV was targeted. The sensitivity of the real-time RT-PCR assay was tested with 10-fold dilutions of viral RNA, and a detection limit of 1 PFU was obtained. The specificity of the assay was demon-strated by testing with other environmental pathogens and indicator microorganisms, and only HAV was positively identified. When combined with immunomagnetic separation, the real-time RT-PCR assay success-fully detected as few as 20 PFU in seeded groundwater samples. Because of its simplicity and specificity, this assay has broad applications for the rapid detection of HAV in contaminated foods or water. Hepatitis A virus (HAV) is an important cause of acute hepatitis worldwide that can lead to severe illness or even death. It is transmitted by the fecal-oral route through the consumption of contaminated foods or water. Traditional methods for HAV detection based on cell culture propagation are often difficult to perform and can take more than 1 week before any visual cytopathic effects are observed (2). Molecula