Simple method of concentrating enteroviruses and hepatitis A virus from sewage and ocean water for rapid detection by reverse transcriptase-polymerase chain reaction.
A rapid and simple method was developed to detect enteroviruses and hepatitis A virus (HAV) in sewage and ocean water. Sewage samples were concentrated by Centriprep-100 and Centricon-100 at 1,000 x g. Samples collected from estuary and near-shore surf zone ocean water in Southern California were concentrated by vortex flow filtration and microconcentration. Reverse transcriptase-polymerase chain reaction (RT-PCR), with enterovirus primers or HAV capsid-specific primers, was used to detect enteroviruses or HAV in all concentrated samples. A nonradioactive internal probe was used to confirm the amplified products. Results of seeding experiments indicated that at 4 degrees C, HAV was more persistent than poliovirus in seawater and both HAV and poliovirus persisted longer at 4 degrees C than at 25 degrees C. RT-PCR was at least 500-fold more sensitive than cell culture. Results were obtained within 5 h by RT-PCR, in contrast with the 5 days to 3 weeks required for cell culture.
Tsai
Y L
YL
Environmental Sciences Laboratory, County Santitation Districts of Orange County, Fountain Valley, California 92728.
Sobsey
M D
MD
Sangermano
L R
LR
Palmer
C J
CJ
D001483:Base Sequence; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics; D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification*; D006507:Hepatovirus / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D017955:Poliovirus / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D012194:RNA-Directed DNA Polymerase; D012623:Seawater; D012722:Sewage; D014871:Water Microbiology
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D012722:Sewage; D012194:RNA-Directed DNA Polymerase