Detection by Duplex Rt-coupled Nested Pcr of Hepatitis a and Rotavirus in Oysters from Thailand East Coast.

Abstract. An efficient and rapid virus detection method is required for routine monitoring and risk assessment in food products. A duplex RT-coupled nested PCR method was developed to detect the simultaneous presence of hepatitis A virus (HAV) and rotavirus in commercial oysters from the eastern coast of Thailand. Primers were designed to amplify HAV VP4 and rotavirus VP7 genes. Although excess amounts of target template of one virus type interfered with RT-PCR am- plification of the other, this was overcome by including a nested duplex PCR step. Detection limit for both types of virus of this technique in oyster samples was more than 1,000-fold lower than that of the equivalent monoplex method. Out of 41 oyster samples 63% were positive for either one or both viruses. All rotaviruses belonged to group A G1P[8]. The use of multiplex RT-coupled nested PCR technique provides a cost-effective, rapid, sensitive and efficient tool to detect a wide diversity of viral pathogens and to improve control of virus infection in oysters.