RAPID MICROBIAL DIAGNOSIS BY NUCLEIC ACID AMPLIFICATION

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A number of factors, especially the prospect of antiviral chemotherapy, have made the need for rapid, sensitive, specific and practical assays for microbial diagnosis increasingly apparent. Progress has been limited in developing rapid viral diagnostic methods largely because of the limited amount of viral analyte molecules (antigen and nucleic acid) in clinical material. The conceptual breakthrough of amplifying an analyte molecule with the polymerase chain reaction has provided new promise for the development of needed assays. The principal investigator and his colleagues have been investigating gene amplification methods to detect viral nucleic acid in clinical material utilizing HIV- I as the prototype agent. Most recently they have been able to detect the sequential appearance of the 4 nucleotide mutations in the reverse transcriptase gene that account for AZT resistance in serial lymphocyte samples from patients receiving AZT therapy. An alternative method for sequence amplification has been developed that in an isothermal (37 degrees C), single cycle reaction that can generate 10(6) copies in 45 minutes. A bead based sandwich capture assay has also been developed that permits reproducible detection and quantitation of the products of this new amplification scheme. This assay is also amenable to nonisotopic detection. A device and method for the simultaneous assay for multiple agents in a single specimen has also been developed. In this proposal methods will be optimized and then applied to four sets of target organisms:herpesviruses, respiratory viruses, genital pathogens and ophthalmologic pathogens. The principal investigator has extensive experience in the clinical diagnosis of antibodies, antigens, and nucleic acids. Large inventories of well characterized clinical specimens for each of the relevant agents are available for these investigations. A systematic, sequential approach will be pursued to fulfill the following aims: AIM 1: Identify nucleic acid sequences from target organisms that will provide organism specificity while maintaining broad reactivity with all strains of that organism. AIM 2: Apply the specific amplification and detection methods to clinical materials known to contain the target agent (and controls) to verify sensitivity, specificity, reproduceability and when possible quantitation. AIM 3: Once assays for individual agents in a panel for the sets of target organisms have been developed, apply the plastic chamber or derivative technologies to develop a rapid diagnostic method for a clinical syndrome (respiratory infection, genital lesions, keratoconjunctivitis).