Norwalk-like (NLVs; human caliciviruses) are important causative agents ofwaterborne gastrointestinal illness. NLVs are not culturable at this time, exceptin humans. The most sensitive and specific methods for detection of NLVs arenucleic acid (NA) amplification and characterization. However, a standardized NAmethod does not exist. To date, at least 10 primer sets, mostly targeting thepolymerase region, have been described for broad amplification and detection ofNLVs, but no single set of primers consistently detects all NLV strains. Asensitive, standardized approach is needed for the detection of NLVs inenvironmental samples. The authors have developed a framework for comparativeevaluation of published, third generation primer sets for NA amplification ofNLV genomes. The framework was developed to provide a consistent set of RT-PCRconditions to compare the primer sets on the basis of sensitivity andreproducibility of amplification on a diversified panel of clinical NLV isolates.This study is a first step in the development of a standardized and robustprotocol for RT-PCR detection on NLVs in water. The ultimate goal of the study isto evaluate several candidate primer sets and amplification conditions, in orderto determine which ones are most sensitive while also giving broad detection ofall relevant NLVs. Includes 13 references, tables.