A comparative amplification of five different genomic regions on Coxsackie A and B viruses. Implications in clinical diagnostics
Modern molecular approaches in Human Enterovirus detection rely on the designing of generic and often degenerate primers in order to amplify specific sequences within the enterovirus genome. In the present study a comparative application of primer sets targeting 5'UTR, the VP1 region, the 3D region as well as a long genomic fragment including the 3'end of VP1, the full length of 2A and 2B, and the 5' moiety of the 2C-coding region was attempted, in order to evaluate their specificity and suitability. The best amplification results from the investigation of 21 CAV reference strains, all six CBV reference strains and 44 clinical strains varying in origin and time of isolation, arose using primer sets 292-222 and UC53-UG52. Based on the above results we conclude that some of the published protocols need to be improved so as to fulfill the demands of an accurate detection and typing of Coxsackie A and B viruses. Contrarily, two of the protocols applied were proved to be more accurate in terms of specificity and general applicability, suggesting that RT-PCR followed by a simple RFLP assay in the case of primer pair UC53-UG52 or by sequencing and sequence analysis in the case of primer set 292-222 should constitute alternative means of modern typing and diagnostics against conventional immunological classification methods. (C) 2004 Elsevier Ltd. All rights reserved.