Simultaneous detection of enteric viruses by multiplex real-time RT-PCR.

A multiplex real-time RT-PCR protocol for the simultaneous detection of noroviruses ("Norwalk-like viruses") of genogroups I and II, human astroviruses and enteroviruses is described. The protocol was developed and evaluated using the LightCycler and corresponding SYBR Green reagents. New primers were designed within conserved genome regions to optimize the detection range of virus subtypes of each genus. To enable the development of a multiplex PCR assay within one tube (capillary), similar mastermix- and cycling-conditions were respected for each individual primer system. Subsequent melting curve analysis allowed the determination of possible dual-contaminations of entero- and noro- or astroviruses by the formation of dual peaks. Special care was taken to minimize the loss of sensitivity, since the detection of small viral contaminations is a crucial parameter especially for food analysis. The multiplex assay was compared successfully to the single SYBR Green assay, and revealed to be at least 10 times more sensitive than the one obtained with an endpoint PCR thermocycler protocol published previously.