title: Evaluation of a degenerate primer for the PCR detection of human caliciviruses.
abstract: Numerous outbreaks of gastroenteritis have been associated with Norwalk virus and Small Round Structured Viruses (SRSVs). These single-stranded RNA viruses, recently classified in the Caliciviridae, have been divided into three genogroups. Antigenic relationships also have been established among the different strains. As both an in vitro culture system and an animal model are lacking for these viruses, virus detection depends primarily on electron microscopy, immunological assays or molecular detection. In this study we first analyzed the genetic homology of the RNA polymerase region for 40 SRSV strains. From a consensus sequence for these strains, we designed a degenerate oligonucleotide to prime cDNA synthesis from viral RNA. We evaluated the degenerate primer in combination with three previously described primers in PCR reactions. A panel of 15 stools containing SRSVs, typed when possible by solid phase immune electron microscopy (SPIEM), were selected to represent all three genogroups and four different SPIEM antigenic types. Serial dilutions of the purified viral nucleic acids were amplified using the three different primer sets. Virus-specific probes were used to characterize the amplicons obtained. Virus-specific amplicons were obtained with at least one primer pair for each strain, but apparent viral RNA titers differed as much as 1000-fold between primer sets. Amplicons from all but one of the 15 strains were confirmed as virus-specific using a panel of 10 different probes. Correlations between the most sensitive primer pair and SPIEM type were seen. This study showed that a single degenerate 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.
authors.affiliation: Division of Molecular Virology, Baylor College of Medicine, Houston, Texas, USA.
authors.forename: F
authors.initials: F
authors.lastname: Le Guyader
authors.forename: M K
authors.initials: MK
authors.lastname: Estes
authors.forename: M E
authors.initials: ME
authors.lastname: Hardy
authors.forename: F H
authors.initials: FH
authors.lastname: Neill
authors.forename: J
authors.initials: J
authors.lastname: Green
authors.forename: D W
authors.initials: DW
authors.lastname: Brown
authors.forename: R L
authors.initials: RL
authors.lastname: Atmar
mesh_terms: D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D005069:Evaluation Studies as Topic; D005243:Feces / Q000821:virology; D006801:Humans; D009663:Norwalk virus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases