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February 1, 1990Journal of Clinical MicrobiologyOpen Access

Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens

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Authors

VGVera GouvêaRGRoger I. GlassPWPatricia Woods

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Overview

Diagnostic study demonstrates rapid PCR amplification and typing of rotavirus VP7 directly from stool specimens, indicating a reliable alternative to monoclonal antibody serotyping.

Key Points

  • To develop and optimize a direct reverse transcription-polymerase chain reaction (PCR) method for amplifying and typing the rotavirus VP7 gene directly from stool specimens without virus culture.
  • Extracted double-stranded RNA directly from stool samples and reference strains (covering human serotypes 1, 2, 3, 4, 8, and 9) for single-tube reverse transcription and Taq polymerase amplification.
  • Optimized MgCl2, dimethyl sulfoxide, and template RNA concentrations, designing serotype-specific primers yielding characteristic segment sizes distinguishable on agarose gels.
  • Evaluated assay performance on 10 rotavirus reference strains, 34 stool specimens previously classified by monoclonal antibody enzyme immunoassay, and 14 previously nonserotypable specimens.
  • Achieved complete (100%) concordance with monoclonal antibody enzyme immunoassay results across all 34 pre-typed stool specimens and correctly identified all 6 human reference serotypes.
  • Successfully determined the specific serotype for 14 stool specimens that could not be typed by standard monoclonal antibody enzyme immunoassays.
  • Generated high yields of VP7 cDNA directly from clinical samples suitable for downstream cloning and sequencing, eliminating the need for cell culture.

Cite This Study

Gouvêa et al. (1990) studied this question.

synapsesocial.com/papers/6a6fb0a7e5469ee92be08e31https://doi.org/10.1128/jcm.28.2.276-282.1990
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Also Consider

Synapse has enriched 4 closely related papers on similar clinical questions. Consider them for comparative context:

  1. 1A candidate for a new serotype of human rotavirus1985 · 221 citations
  2. 2Rotavirus isolate WI61 representing a presumptive new human serotype1987 · 242 citations
  3. 3Production and preliminary characterization of monoclonal antibodies directed at two surface proteins of rhesus rotavirus1983 · 287 citations
  4. 4Prediction of human rotavirus serotype by nucleotide sequence analysis of the VP7 protein gene1988 · 117 citations