Tansuphasiri U, Vathanophas K, Pariyanonda A, Kittigul L, Utrarachkij F, Diraphat P, Siripanichgon K, Punchitton S, Chitpirom K, Cheaochantanakij N
Department of Microbiology, Faculty of Public Health, Mahidol University, Bangkok, Thailand.
Southeast Asian J Trop Med Public Health. 2000 Mar;31(1):47-56.
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%.
本研究描述了一种通过简单的逆转录聚合酶链反应（RT-PCR）在环境水体中快速检测脊髓灰质炎病毒的方法。该方法使用两对引物，通过一步法在单个反应中区分脊髓灰质炎病毒和非脊髓灰质炎肠道病毒，将逆转录和PCR结合在一个管中。通过琼脂糖凝胶电泳检测，检测的脊髓灰质炎病毒产生了两条分别为153 bp和293 bp的条带，无需进一步杂交。用热处理从感染细胞提取物上清中提取的RNA测量，这种一步双RT-PCR的检测灵敏度为10（-1）50%组织培养感染剂量（TCID50）。该检测方法用于评估基于膜滤吸附和洗脱的样品浓缩能力，以及基于异硫氰酸胍-苯酚-氯仿提取的简单RNA分离纯化能力；该系统对接种于5升自来水中的病毒的检测限为5×10（-1）TCID50。该方案应用于1998年2月和5月从泰国曼谷2个社区采集的环境水样中的脊髓灰质炎病毒检测。在检测的100个样品中，从同一地点的一条开放污水管道采集的2个水样脊髓灰质炎病毒呈阳性，从另一条污水管道采集的1个样品非脊髓灰质炎肠道病毒呈阳性，另外97个水样脊髓灰质炎病毒和非脊髓灰质炎肠道病毒均为阴性。采用细胞培养技术检测脊髓灰质炎病毒，检测的100个样品中1型、2型或3型脊髓灰质炎病毒均未呈阳性。RT-PCR比细胞培养技术更灵敏、快速、简单且具有成本效益，因为通过RT-PCR呈脊髓灰质炎病毒阳性的2个水样未能通过细胞培养检测到。将阳性样品293 bp扩增子的序列数据与Genbank和EMBL数据库中参考脊髓灰质炎病毒株的序列数据进行比较，发现与1型Sabin株序列的同一性为99%。
