We compared primers rather than assay function using clinical material instead of cultured virus, plasmid or synthetic RNA standards, or screening contemporary or archived extracts, which are sometimes of low viral load. When picornavirus epidemiology is the primary research focus, we recommend using >2 primer pairs to maximize the detection of HRVs. Under our conditions, pairs 1-4 returned the highest number of positive results, and the rtP-CRs behaved similarly but with reduced sensitivity. The rtPCR that used pair 5 did not amplify known HEVs.