SOLID PHASE PLATE ASSAY (SPPA) FOR HIV1 GENOTYPING

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A nucleic acid based solid phase microplate capture assay is being developed for the differentiation of the major HIV-1 subtypes of groups M and O. Sample processing conditions is optimized to ensure the assay's sensitivity and reproducibility. Ah internal control vector containing a synthetic fragment flanked by sequences corresponding to the HIV primers are designed to monitor sample recovery during extraction, reverse transcription, PCR amplification and detection. Biotin- labeled degenerate primers from the conserved region of HIV-1 env are selected such that all known HIV-1 isolates, including type O, could be amplified. The labeled RT-PCR amplicons containing the hyper-variable region of env are captured on separate microtiter wells by hybridization to specific immobilized capture oligomers representing each of the designated subtypes, and colorimetrically detected by a chromogenic reaction using a plate reader. Several dozen of HIV- 1 viral isolates collected from different geographic regions are tested and subtype designations are confirmed by direct sequencing of viral gag and env regions. This newly developed microplate capture assay can detect and differentiate all major subtypes of HIV-1, including subtypes A, B, C, D, E, F and HIV-1 subtype O. This genotyping assay can be used as a sensitive and robust screening and genotyping method for monitoring the extent of global HIV-1 variation and for disease management. PROPOSED COMMERCIAL APPLICATION: The assay will provide a method for HIV-l subtyping of clinical samples and for the production of molecular controls and kits useful for developers of candidate vaccines and antivirals, to physicians in the assessment of anti- retroviral treatment and drug resistance of HIV-l strains, and to scientists and clinical laboratories to evaluate the performance of HIV-l detection assays. Manufacturers of microdevices, microchips and automated systems could adapt this assay into a biochip format to create a HIV-l genotyping system.