DETECTION OF POLIOVIRUS, HEPATITIS-A VIRUS, AND ROTAVIRUS FROM SEWAGEAND OCEAN WATER BY TRIPLER REVERSE-TRANSCRIPTASE PCR

A tripler reverse transcriptase PCR (RT-PCR) was developed to simultan eously detect poliovirus, hepatitis A virus (HAV), and rotavirus in se wage and ocean water. Sewage and ocean water samples seeded with the t hree different viruses were concentrated by ultrafiltration. The unsee ded ocean water and sewage samples were concentrated by vortex flow fi ltration and/or ultrafiltration. Random hexamers and a rotavirus downs tream primer were used to initiate reverse transcription. Three differ ent sets of primers specific for poliovirus, HAV, and rotavirus cDNAs were mixed in the PCR mixture to amplify the target DNA. Three distinc t amplified DNA products representing poliovirus, HAV, and rotavirus w ere identified by gel electrophoresis as 394-, 192-, and 278-bp sequen ces, respectively. Dot blot and Southern analyses were used to confirm the amplified products for each virus present in the environmental sa mples. Except for poliovirus, the sensitivity of tripler RT-PCR for th e detection of rotavirus and HAV was found to be similar to that of mo noplex RT-PCR, which uses only one set of primers to amplify a single type of virus. The tripler RT-PCR has greater advantages over monoplex RT-PCR for virus detection, namely, the rapid turnaround time and cos t effectiveness.