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 10(5)-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 microliters (0.2 PFU/l tap water).
Ma
J F
JF
Department of Microbiology and Immunology, University of Arizona, Tucson 85721, USA.
Gerba
C P
CP
Pepper
I L
IL
D006801:Humans; D017955:Poliovirus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis; D012194:RNA-Directed DNA Polymerase; D012680:Sensitivity and Specificity; D014871:Water Microbiology; D014881:Water Supply
D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase