A simple device for the concentration and detection of enterovirus, hepatitis E virus and rotavirus from water samples by reverse transcription-polymerase chain reaction.
A simultaneous concentration of enteroviruses, hepatitis E virus, and rotavirus from drinking water samples through a filtration column filled with granular activated carbon (GAC) was achieved. Urea-arginine phosphate buffer (UAPB) as an eluent at pH 9.0 was used for effective desorption and elution of viruses from GAC. Further concentration of viruses with magnesium chloride enabled nucleic acid extraction, cDNA synthesis, amplification with a specific set of primers for enterovirus, hepatitis E virus and rotavirus. Polymerase chain reaction (PCR) products were then confirmed by Southern transfer and hybridization with the relevant probes. The efficacy of the protocol was established with 100 1 of water samples seeded with poliovirus-1, providing 74% recovery in granular activated carbon based UAPB-RT-PCR. The GAC-based method for concentration of viruses from water samples was preferred, despite its somewhat lower efficacy compared to 80% in membrane filter based UAPB-RT-PCR protocol, due to the specific requirements of short-time and savings in cost of analyses. The protocol was used for the detection of waterborne viruses from 24 drinking water sources in urban areas of New Delhi. Direct isolation of viruses from water samples revealed that the 4 samples were positive for enteroviruses, two for hepatitis E virus, and 10 samples for rotavirus. One sample was positive for both hepatitis E virus and rotavirus, and another for all the 3 types of viruses.
Jothikumar
N
N
National Environmental Engineering Research Institute (NEERI), Madras Zonal Laboratory, India. vsnb@mcimail.com
Khanna
P
P
Paulmurugan
R
R
Kamatchiammal
S
S
Padmanabhan
P
P
D000818:Animals; D001483:Base Sequence; D002021:Buffers; D002460:Cell Line; D002522:Chlorocebus aethiops; D004279:DNA, Viral; D016752:Hepatitis E virus / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007194:India; D008969:Molecular Sequence Data; D017955:Poliovirus / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012401:Rotavirus / Q000235:genetics / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic; D014871:Water Microbiology; D014881:Water Supply
D002021:Buffers; D004279:DNA, Viral