Publication: Rapid detection of polioviruses in environmental water samples by one-step duplex RT-PCR
Issued Date
2000-03-01
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ISSN
01251562
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2-s2.0-0034156994
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Mahidol University
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SCOPUS
Bibliographic Citation
Southeast Asian Journal of Tropical Medicine and Public Health. Vol.31, No.1 (2000), 47-56
Suggested Citation
Unchalee Tansuphasiri, Kanda Vathanophas, Anong Pariyanonda, Leera Kittigul, Fuangfa Utrarachkij, Pornphan Diraphat, Kanokrat Siripanichgon, Supornvit Punchitton, Kitja Chitpirom, Nattasai Cheaochantanakij Rapid detection of polioviruses in environmental water samples by one-step duplex RT-PCR. Southeast Asian Journal of Tropical Medicine and Public Health. Vol.31, No.1 (2000), 47-56. Retrieved from: https://repository.li.mahidol.ac.th/handle/123456789/26265
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Title
Rapid detection of polioviruses in environmental water samples by one-step duplex RT-PCR
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Abstract
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 pcliovirus 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 × 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 2.93-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%.