Rapid detection of polioviruses in environmental water samples by one-step duplex RT-PCR.
This study describes the rapid detection of polioviruses in environmental waters by a simple reverse transcriptase-polymerase chain reaction (RT-PCR) using two primer pairs for differentiation of poliovirus from non-polio enteroviruses in a single reaction by a one-step method, combining RT and PCR in a single tube. The detection by agarose gel electrophoresis yielded 2 bands of 153-bp and 293-bp for poliovirus tested without the need for further hybridization. The detection sensitivity of this one-step duplex RT-PCR, as measured with RNA extracted by heat treatment from supernatant of infected cell extracts, was 10(-1) 50% tissue culture effective doses (TCID50). This assay was used to evaluate the ability of sample concentration by membrane filter-based adsorption and elution, and purification by a simple RNA isolation based on guanidine isothiocyanate-phenol-chloroform extraction; the system yielded a detection limit of 5 x 10(-1) TCID50 seeded in 5 liters of tap water. This protocol was applied to the poliovirus detection in environmental water collected from 2 communities in Bangkok, Thailand during February and May 1998. Of 100 samples tested, 2 water samples collected from the same open sewage pipeline at one location were positive for polioviruses and one sample collected from another sewage pipeline was positive for non-polio enterovirus while a further 97 water samples were negative for both polioviruses and non-polio enteroviruses. With poliovirus detection by cell culture technique, none of the 100 samples tested was positive for poliovirus type 1, 2 or 3. RT-PCR was more sensitive, rapid, simple and cost-effective than the cell culture technique since the two water samples which were positive for polioviruses by RT-PCR failed to be detected by cell culture. Sequence data of 293-bp amplicons from positive samples were compared with those of reference poliovirus strains in the Genbank and the EMBL databases and identity to the sequence of type 1 strain Sabin was found to be 99%.
Tansuphasiri
U
U
Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, Thailand.
Vathanophas
K
K
Pariyanonda
A
A
Kittigul
L
L
Utrarachkij
F
F
Diraphat
P
P
Siripanichgon
K
K
Punchitton
S
S
Chitpirom
K
K
Cheaochantanakij
N
N
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D004587:Electrophoresis, Agar Gel; D005374:Filtration; D017955:Poliovirus / Q000235:genetics / Q000302:isolation & purification*; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012722:Sewage / Q000821:virology*; D013785:Thailand; D014776:Virus Cultivation; D014871:Water Microbiology
D012367:RNA, Viral; D012722:Sewage