Low cost molecular assay for HIV

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DESCRIPTION (provided by applicant): We propose to develop a rapid, low cost, threshold assay for the detection of HIV infection of HIV vaccination trial participants. The assay we propose to develop will use our proprietary helicase dependent amplification (HDA) platform as well as a low cost device specifically designed to perform molecular tests without contaminating the laboratory with amplification products. HDA is similar to the polymerase chain reaction (PCR) in that it uses two primers to exponentially amplify nucleic acids. It is distinct from PCR in that it is entirely isothermal (and thus does not require costly thermocyclers). We propose to use a lateral flow device to detect amplification products. Such devices have already proven their utility in separating nucleic acid amplification products, and are widely used in several "moderate complexity" and CLIA-waived tests listed in the FDA device database. Detection of amplicons will be accomplished by using latex particles conjugated to antibodies that bind to the probes used to detect the HDA reaction products. The assay uses a sandwich format to detect haptens (biotin, FITC and Digoxigenin) incorporated into one of the HDA primers and into each of the detection probes. The lateral flow strip used for these assays has two capture zones, and thus allows for the detection of one analyte (HIV) as well as a competitive internal control. Assays that fail to give a band in the lateral flow device window are scored as invalid, while assays with a band in the control line alone are scored as true negative. Assays with bands at both the control and test line position are scored as positives at the threshold value (typically 200 copies of the analyte). Assays with a strong band in the test line but no control line are scored as strong positives (typically over 5000 copies of analyte). Preliminary studies suggest a limit of detection of 100 copies/mL plasma when nucleic acids extracted from plasma samples with a sequence-specific capture method are used as templates for RT-HDA. We propose to further test the specificity and sensitivity of the HIV test with clinical specimens obtained from 5 high-risk patient recruitment sites. Assays will be performed at BioHelix, and at Vanderbilt University. Kits will be supplied to Dr. Yi-Wei Tang (Vanderbilt University) so his laboratory can evaluate the assays. In addition, we propose to perform a validation study at the National Institute for Communicable Diseases (NICD) of South Africa to gain clearance to sell kits in South Africa. Finally, we may also contract with Assuragen to engineer a clone of Armored RNA for our competitive internal control. By the end of Phase II, we will have preliminary data for submission of an IDE to the FDA to kick-off a clinical study to seek pre market approval (PMA) for sale of the assay system for human diagnostics in the US.