Sensitive detection of group A rotaviruses by immunomagnetic separation and reverse transcription-polymerase chain reaction

Abstract

An immunomagnetic separation (IMS) method was developed for concentrating rotaviruses from environmental samples, as well as a reverse transcription-polymerase chain reaction (RT-PCR) for the sensitive and specific detection of group A rotaviruses. Magnetic beads were coated with monoclonal antibodies directed against the group-specific, inner capsid protein (VP6) and subsequently used to capture and purify the virus with the help of a magnet. The genome was made available for RT by heat-disrupting the viral particles. A single 40-cycle PCR was as sensitive as a nested PCR, both detecting 0.005 PFU of the Wa strain, corresponding to approximately 5 particles as indicated by EM. The nested PCR was positive for all the group A strains tested, but negative for group C rotaviruses and other RNA viruses. The IMS-RT-PCR method functioned satisfactorily with virus seeded out in fresh water samples; with sea water, the IMS removed most, but not all, inhibiting activity.