title: Comparison of enzyme immunoassay, PCR, and type-specific cDNA probe techniques for identification of group A rotavirus gene 4 types (P types).
abstract: This study was designed to evaluate three techniques most commonly used to identify the VP4 (P) types of human group A fecal rotaviruses. The techniques included PCR with nested primers and hybridization with PCR-generated probes (to determine the P genotypes). The results obtained by these genetic techniques were evaluated against those obtained by an enzyme immunoassay (EIA) incorporating neutralizing monoclonal antibodies (N-MAbs) reacting with three major human P serotypes (serotypes P1A, P1B, and P2A). The P types of the rotaviruses present in 102 fecal specimens were determined under code by each of the three assays. The specificity of each assay was evaluated against a "gold standard" putative P type (P serotype and genotype) deduced from knowledge of the VP7 (G) type and the origin of the fecal specimen. Overall comparison of the results showed respective sensitivities and specificities of 92 and 92% for reverse transcription-PCR, 80 and 99% for hybridization, and 73 and 91% for EIA with N-MAbs. The hybridization assay retained high sensitivity with specimens stored for > or = 10 years. Hybridization assays with nonradioactive probes are relatively inexpensive and are suited for use in developing countries. In summary, both genetic assays showed high sensitivities and specificities in assigning a P type to human fecal rotavirus strains. Further evaluation of the EIA with N-MAbs is required, together with incorporation of new N-MAbs for the detection of the additional P types detected in developing countries.
authors.affiliation: Department of Gastroenterology and Clinical Nutrition, Royal Children's Hospital, Melbourne, Victoria, Australia.
authors.forename: P J
authors.initials: PJ
authors.lastname: Masendycz
authors.forename: E A
authors.initials: EA
authors.lastname: Palombo
authors.forename: R J
authors.initials: RJ
authors.lastname: Gorrell
authors.forename: R F
authors.initials: RF
authors.lastname: Bishop
mesh_terms: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary / Q000235:genetics; D003967:Diarrhea / Q000821:virology; D005069:Evaluation Studies as Topic; D005814:Genes, Viral; D006801:Humans; D007124:Immunoenzyme Techniques / Q000706:statistics & numerical data; D015336:Molecular Probe Techniques / Q000706:statistics & numerical data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012401:Rotavirus / Q000145:classification* / Q000235:genetics* / Q000276:immunology; D012400:Rotavirus Infections / Q000821:virology; D012680:Sensitivity and Specificity; D012703:Serotyping
chemical_list: D000911:Antibodies, Monoclonal; D018076:DNA, Complementary