Simultaneous detection and identification of hepatitis A virus and rotavirus by multiplex nucleic acid sequence-based amplification (NASBA) and microtiter plate hybridization system.

Human rotavirus and hepatitis A virus (HAV) are two of the most common causes of virus-mediated food-borne illness. Epidemiological investigations of outbreaks associated with these viruses have been hindered by the lack of available methods for their detection in foodstuffs. In this study, a multiplex nucleic acid sequence-based amplification (NASBA) system was developed to detect specifically and simultaneously human rotavirus and HAV. Two sets of primers selected from published nucleic acid sequences were used in the NASBA mixture to amplify viral RNA from both viruses. Denaturing gel electrophoresis revealed two distinct RNA products with 268 and 474 nucleotides amplified from rotavirus and HAV, respectively. The specificity of the multiplex NASBA was confirmed by a microtiter plate hybridization and detection system and by Northern blot analysis using specific oligonucleotide probes. The presence of non-homologous nucleic acid and non-target microorganisms did not have any effect on the specificity of the multiplex NASBA. Using the optimized NASBA and microtiter plate hybridization conditions, as little as 400 PFU ml x (-1) of HAV and 40 PFU ml x (-1) of rotavirus were detected. The multiplex NASBA system offers advantages over monoplex virus detection systems in terms of turnaround time and cost-effectiveness.