Nucleic acid hybridization is a promising technique for the rapid and sensitive diagnosis of microbial agents in body fluids. These techniques, as currently applied to clinical diagnostic medicine, suffer from two serious limitations. Reported clinical studies reveal a sensitivity compared to standard assays of only 50-80% and the use of isotopically labelled probes poses a significant biohazard. We propose to develop affinity labelled probes by linking nucleic acid to a protein molecule whic is labelled with the co-factor biotin. The specific, rapid and tenacious interaction of biotin with avidin provides the bases for constructing more sensitive enzymatic detection methods without the hazards of radioisotopes. Standard dot and Southern transfer hybridization assays on nitrocellulose as well as sandwich hybridization assays will be developed utilizing DNA-protein conjugates as probes. In addition, we will explore alternative solid supports, including plastics and Sephacryl S or Sepharose CL, to achieve the greater sensitivity and objectivity obtainable with soluble substrates for the enzyme detection method. The sensitivity and specificity of hybridization will be compared with established diagnostic methods for the detection of enteric pathogens in stool specimens. The enteric pathogens to be studied include rotavirus, adenovirus, Giardia lamblia and Escheria coli expressing the heat stable or labile toxin. In a final phase of the research plan we will prospectively study the sensitivity, specificity and clinical utility of hybridization for the diagnosis of the above mentioned enteric pathogens in children attending day-care centers, residents of nursing homes, hospitalized children and recipients of bone-marrow transplants. These clinical populations are frequently exposed to transmissible agents of diarrheal disease and rapid sensitive diagnostic tests would greatly improve the prospects for the prevention and control of enteric illness in these patients. In support of the research plan, I will pursue a Phase I program of laboratory and academic studies to further my knowledge of nucleic acid biochemistry, applied molecular biology, enzyme detection systems and basic biostatistical and epidemiology methods.
|Coutlee, F; Rubalcaba, E A; Viscidi, R P et al. (1990) Quantitative detection of messenger RNA by solution hybridization and enzyme immunoassay. J Biol Chem 265:11601-4|
|Newman, C L; Modlin, J; Yolken, R H et al. (1989) Solution hybridization and enzyme immunoassay for biotinylated DNA-RNA hybrids to detect enteroviral RNA in cell culture. Mol Cell Probes 3:375-82|
|Coutlee, F; Yolken, R H; Viscidi, R P (1989) Nonisotopic detection of RNA in an enzyme immunoassay using a monoclonal antibody against DNA-RNA hybrids. Anal Biochem 181:153-62|
|Coutlee, F; Bobo, L; Mayur, K et al. (1989) Immunodetection of DNA with biotinylated RNA probes: a study of reactivity of a monoclonal antibody to DNA-RNA hybrids. Anal Biochem 181:96-105|
|Coutlee, F; Viscidi, R P; Yolken, R H (1989) Comparison of colorimetric, fluorescent, and enzymatic amplification substrate systems in an enzyme immunoassay for detection of DNA-RNA hybrids. J Clin Microbiol 27:1002-7|
|Viscidi, R P; O'Meara, C; Farzadegan, H et al. (1989) Monoclonal antibody solution hybridization assay for detection of human immunodeficiency virus nucleic acids. J Clin Microbiol 27:120-5|
|Viscidi, R P; Connelly, C J; Yolken, R H (1986) Novel chemical method for the preparation of nucleic acids for nonisotopic hybridization. J Clin Microbiol 23:311-7|