Antigen Capture/Polymerase Chain Reaction for the Detection of Hepatitis A Virus in Human Clinical Materials
Human hepatitis A virus (HAV) is the single most common cause of acute viral hepatitis in many regions of the world. Recently classified within the genus Hepatovirus of the family Picornaviridae, this positive-strand RNA virus has several notable biological features. Unlike many picornaviruses, HAV has a protracted, most often noncytolytic replication cycle in cell culture which makes the isolation of this virus from clinical materials exceptionally difficult. To facilitate the detection of HAV in clinical samples and to allow us to determine the genetic relatedness of epidemiologically unrelated virus strains, we have developed an antigen capture/polymerase chain reaction (AC/PCR) method which utilizes a simple immunoaffmity-based virus purification scheme prior to isolation of viral RNA, reverse transcription, and PCR (Jansen et al. 1990). This method involves the “capture” of virus by a monoclonal antibody bound to a solid-phase support (the PCR reaction tube itself), heat denaturation of the virus which results in release of its RNA, and reverse transcription followed immediately by PCR. Only a single reaction tube is used throughout the entire process, and thus the method involves fewer manipulations than conventional PCR methods for detection of RNA viruses. In addition, the immunoaffmity virus purification step results in a greatly increased abundance of the target RNA relative to extraneous nucleic acids subjected to PCR. The specificity is thus enhanced, and AC/PCR usually generates a single PCR product which is readily visualized on agarose gels containing ethidium bromide. As little as 5 μl of a clinical sample (usually a crude fecal suspension) is required. The general method and its application to the study of the molecular epidemiology of HAV have been described in detail elsewhere (Jansen et al. 1990).
KeywordsFecal Suspension Single Reaction Tube Viral Nucleic Acid Sequence Differential Signal Strength Additional Phosphate Buffer Saline
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