A multiplex reverse transcription polymerase chain reaction method for thedetection of foodborne viruses

A multiplex reverse transcription polymerase chain reaction (RT-PCR) method was developed for the simultaneous detection of the human enteroviruses, h epatitis A virus (HAV) and Norwalk virus (NV). Poliovirus type 1 (PV1) was chosen as a model for the human enterovirus group. Three different sets of primers were used to produce three size-specific amplicons of 435 bp, 270 b p, and 192 bp for PV1, NV, and HAV, respectively. RT-PCR products were sepa rated by agarose gel electrophoresis, and amplicon identity was confirmed b y Southern transfer followed by DNA hybridization using nonradioactive, dig oxigenin-labeled internal probes. When tested an mixed, purified virus susp ensions, the multiplex method achieved detection limits of less than or equ al to 1 infectious unit (PV1 and HAV) or RT-PCR-amplifiable unit (NV) for a ll viruses. With further streamlining efforts such as single tube amplifica tion and liquid hybridization, multiplex PCR offers advantages over cell cu lture methodology and monoplex PCR because it allows for rapid and cast-eff ective detection of several human enteric viruses in a single reaction tube .