EVALUATION OF A DEGENERATE PRIMER FOR THE PCR DETECTION OF HUMAN CALICIVIRUSES - BRIEF REPORT

Numerous outbreaks of gastroenteritis have been associated with Norwal k virus and Small Round Structured Viruses (SRSVs). These single-stran ded RNA viruses, recently classified in the Caliciviridae, have been d ivided into three genogroups. Antigenic relationships also have been e stablished among the different strains. As both an in vitro culture sy stem and an animal model are lacking for these viruses, virus detectio n depends primarily on electron microscopy, immunological assays or mo lecular detection. In this study we first analyzed the genetic homolog y of the RNA polymerase region for 40 SRSV strains. From a consensus s equence for these strains, we designed a degenerate oligonucleotide to prime cDNA synthesis from viral RNA. We evaluated the degenerate prim er in combination with three previously described primers in PCR react ions. A panel of 15 stools containing SRSVs, typed when possible by so lid phase immune electron microscopy (SPIEM), were selected to represe nt all three genogroups and four different SPIEM antigenic types. Seri al dilutions of the purified viral nucleic acids were amplified using the three different primer sets. Virus-specific probes were used to ch aracterize the amplicons obtained. Virus-specific amplicons were obtai ned with at least one primer pair for each strain, but apparent viral RNA titers differed as much as 1000-fold between primer sets. Amplicon s from all but one of the 15 strains were confirmed as virus-specific using a panel of 10 different probes. Correlations between the most pr imer pair and SPIEM type were seen. This study showed that a single de generate primer could be used in cDNA synthesis for a variety of SRSVs but that the sensitivity of the RT-PCR assay depended upon the second primer and virus-specific probes used.