Detection of poliovirus, hepatitis A virus, and rotavirus from sewage and ocean water by triplex reverse transcriptase PCR

Abstract

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.