Sensitive detection of group A rotaviruses by immunomagnetic separation and reverse transcription-polymerase chain reaction.
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.
Grinde
B
B
Department of Virology, National Institute of Public Health, Oslo, Norway.
Jonassen
T O
TO
Ushijima
H
H
D000956:Antigens, Viral / Q000276:immunology; D001483:Base Sequence; D002213:Capsid / Q000235:genetics / Q000276:immunology; D036022:Capsid Proteins; D004279:DNA, Viral / Q000235:genetics; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D000956:Antigens, Viral; D036022:Capsid Proteins; D004279:DNA, Viral; C061332:VP6 protein, Rotavirus