Increased sensitivity of poliovirus detection in tap water concentrates by reverse transcriptase-polymerase chain reaction.
This study developed a methodology to increase the sensitivity of enteric virus detection in tap water concentrates.
Polymerase chain reaction (PCR) detection of virus in reduced volumes of virus-containing water concentrates was successful following removal of PCR inhibitory substances.
Poliovirus 1 and coxsackievirus B3 were seeded into 378 l of tap water, concentrated with 1MDS filters, and reconcentrated by organic flocculation.
The volume of concentrates was successfully reduced from 25 to 5 ml without loss of virus recovery.
PCR detection of virus after treatment of a water concentrate (1.1 X 105-fold concentration) with a Sephadex G-100 plus Chelex-100 column, or Sephadex G-50 plus Chelex-100 column, followed by heat treatment to release viral RNA, was compared with direct phenol-chloroform-isoamyl alcohol (PCI) extraction of viral RNA.
The Sephadex G-50 plus Chelex-100 column did not remove inhibitory substances efficiently.
The Sephadex G-100 plus Chelex-100 column could remove inhibitory substances, however, 99% of the viruses were also removed by the column.
PCI extraction was found to be sufficient to remove inhibitory substances for reverse transcriptase (RT) - seminested PCR with a sensitivity of 0.2 plaque-forming units/10 mul (0.2 PFU/l tap water).
Mots-clés Pascal : Poliovirus, Eau distribution, Concentrat, Détection, Méthode, Réaction chaîne polymérase, Transcription inverse, Enterovirus, Picornaviridae, Virus
Mots-clés Pascal anglais : Poliovirus, Tap water, Concentrate, Detection, Method, Polymerase chain reaction, Reverse transcription, Enterovirus, Picornaviridae, Virus
Notice produite par :
Inist-CNRS - Institut de l'Information Scientifique et Technique
Cote : 96-0071905
Code Inist : 002A05C09. Création : 199608.