Detection of enteroviruses in groundwater with the polymerase chain reaction.
Standard methods for the detection of enteroviruses in environmental samples involve the use of cell culture, which is expensive and time-consuming. The polymerase chain reaction (PCR) is an attractive method for the detection of enteroviruses in water because primary cell culture is not needed and the increased sensitivity of PCR allows detection of the low numbers of target DNAs and RNAs usually found in environmental samples. However, environmental samples often contain substances that inhibit PCR amplification of target DNA and RNA. Procedures that remove substances that interfere with the amplification process need to be developed if PCR is to be successfully applied to environmental samples. An RNA-PCR assay for the detection of enteroviruses in water was developed and used to test a variety of groundwater concentrates and humic acid solutions seeded with poliovirus type 1. The groundwater samples and humic acid solutions were treated with Sephadex G-50, Sephadex G-100, Sephadex G-200, Chelex-100 resin, and a mixed bed resin to remove PCR-inhibitory material from the samples. Sephadex G-100 in combination with Chelex-100 was found to be very effective in removing inhibitory factors for the detection of enteroviruses in groundwater concentrates by PCR. Viruses were detected in two of the groundwater concentrates by the RNA-PCR assay after treatment with Sephadex G-100 plus Chelex-100. This was confirmed by tissue culture, suggesting that the treatment protocol and, subsequently, the RNA-PCR assay are applicable for the detection of enteroviruses in environmental samples.
Abbaszadegan
M
M
Department of Soil and Water Science, University of Arizona, Tucson 85721.
Huber
M S
MS
Gerba
C P
CP
Pepper
I L
IL
D001483:Base Sequence; D002614:Chelating Agents; D004279:DNA, Viral / Q000235:genetics; D003911:Dextrans; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012117:Resins, Synthetic; D012680:Sensitivity and Specificity; D014871:Water Microbiology
D002614:Chelating Agents; D004279:DNA, Viral; D003911:Dextrans; D012117:Resins, Synthetic; C024997:Chelex 100; C025614:sephadex