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.
Mots-clés Pascal : Rotavirus, Reoviridae, Virus, Détection, Méthode, Transcription inverse, Réaction chaîne polymérase, Evaluation performance, Anticorps monoclonal, Méthode immunomagnétique
Mots-clés Pascal anglais : Rotavirus, Reoviridae, Virus, Detection, Method, Reverse transcription, Polymerase chain reaction, Performance evaluation, Monoclonal antibody, Immunomagnetic method
Notice produite par :
Inist-CNRS - Institut de l'Information Scientifique et Technique
Cote : 96-0071908
Code Inist : 002A05C09. Création : 199608.