SENSITIVE DNA HYBRIDIZATION TEST FOR ROTAVIRUS RNA

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Rotaviruses are the major cause of serious infantile diarrhea. This pathogen accounts for 40-60% of the diagnosed cases of gastroenteritis in children under the age of two years. Diagnosis is currently made by ELISA or by electron microscopy. The latter is impractical for the small laboratory, and the ELISA produces an unacceptable number of false positives. The virus consists of 11 segments of double-stranded RNA. The nucleotide sequence of the major outer capsid glycoprotein gene is known for three human serotypes. We propose to synthesize DNA probes which will hybridize specifically with a region of the genome common to the human serotypes. Phase I. Based on published sequence information, we will synthesize an oligonucleotide probe complementary both to the plus strand RNA of the genome and the mRNA of the virus. The probe will be tested for its ability to detect rotavirus in characterized stool samples. The major technical innovation in this work will be the development of methods which allow oligonucleotide probes to be used to detect double-stranded RNA viruses in clinical samples. Phase II. We will conduct clinical trials of the best probes synthesized, primarily non-isotopically labeled in order to produce a clinical diagnostic kit to rapidly detect rotavirus in stool samples.