Tsai Y L, Tran B, Sangermano L R, Palmer C J
Environmental Sciences Laboratory, Fountain Valley, California 92728.
Appl Environ Microbiol. 1994 Jul;60(7):2400-7. doi: 10.1128/aem.60.7.2400-2407.1994.
A triplex reverse transcriptase PCR (RT-PCR) was developed to simultaneously detect poliovirus, hepatitis A virus (HAV), and rotavirus in sewage and ocean water. Sewage and ocean water samples seeded with the three different viruses were concentrated by ultrafiltration. The unseeded ocean water and sewage samples were concentrated by vortex flow filtration and/or ultrafiltration. Random hexamers and a rotavirus downstream primer were used to initiate reverse transcription. Three different sets of primers specific for poliovirus, HAV, and rotavirus cDNAs were mixed in the PCR mixture to amplify the target DNA. Three distinct amplified DNA products representing poliovirus, HAV, and rotavirus were identified by gel electrophoresis as 394-, 192-, and 278-bp sequences, respectively. Dot blot and Southern analyses were used to confirm the amplified products for each virus present in the environmental samples. Except for poliovirus, the sensitivity of triplex RT-PCR for the detection of rotavirus and HAV was found to be similar to that of monoplex RT-PCR, which uses only one set of primers to amplify a single type of virus. The triplex RT-PCR has greater advantages over monoplex RT-PCR for virus detection, namely, the rapid turnaround time and cost effectiveness.
开发了一种三重逆转录聚合酶链反应（RT-PCR），用于同时检测污水和海水中的脊髓灰质炎病毒、甲型肝炎病毒（HAV）和轮状病毒。接种了三种不同病毒的污水和海水样本通过超滤进行浓缩。未接种的海水和污水样本通过涡旋流过滤和/或超滤进行浓缩。使用随机六聚体和轮状病毒下游引物启动逆转录。将针对脊髓灰质炎病毒、甲型肝炎病毒和轮状病毒cDNA的三组不同引物混合在PCR混合物中，以扩增目标DNA。通过凝胶电泳鉴定出分别代表脊髓灰质炎病毒、甲型肝炎病毒和轮状病毒的三种不同扩增DNA产物，其序列长度分别为394、192和278 bp。使用斑点印迹和Southern分析来确认环境样本中每种病毒的扩增产物。除脊髓灰质炎病毒外，发现三重RT-PCR检测轮状病毒和甲型肝炎病毒的灵敏度与单重RT-PCR相似，单重RT-PCR仅使用一组引物扩增单一类型的病毒。在病毒检测方面，三重RT-PCR比单重RT-PCR具有更大的优势，即周转时间快和成本效益高。
