SENSITIVE DETECTION OF GROUP-A ROTAVIRUSES BY IMMUNOMAGNETIC SEPARATION AND REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION

An immunomagnetic separation (IMS) method was developed for concentrat ing rotaviruses from environmental samples, as well as a reverse trans cription-polymerase chain reaction (RT-PCR) for the sensitive and spec ific detection of group A rotaviruses. Magnetic beads were coated with monoclonal antibodies directed against the group-specific, inner caps id 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 he at-disrupting the viral particles. A single 40-cycle PCR was as sensit ive as a nested PCR, both detecting 0.005 PFU of the Wa strain, corres ponding to approximately 5 particles as indicated by EM. The nested PC R was positive for all the group A strains tested, but negative for gr oup C rotaviruses and other RNA viruses. The IMS-RT-PCR method functio ned satisfactorily with virus seeded out in fresh water samples; with sea water, the IMS removed most, but not all, inhibiting activity.