A PCR based HIV-1 test for point-of-care application

HIV-1 proviral DNA detection by PCR is the gold standard test for early diagnosis in infants. In resource limited settings, dried blood samples are transported from the point-of-care (POC) to central laboratories for testing because the test requires complex equipment and skilled personnel. Unfortunately, transport problems frequently prevent result delivery to the physician/patient at the POC leading to high loss to follow up. We have developed a very simple, rapid and accurate real-time PCR based test for POC testing. The method employs a novel DNA extraction technology that uses single-use disposables to extract genomic DNA from blood in less than 5 minutes. The DNA is then analyzed by a multiplexed real-time PCR assay that has an exogenous internal control that reports on PCR inhibition in addition to the primers and probes for HIV-1 detection. Our method demonstrated 97% clinical sensitivity and 100% specificity when tested with infant blood specimens. The method is capable of detecting 5 HIV-1 copies in 100microl of blood. The method also allows for storage of blood samples for at least one month after blood collection. In order to make the test field-deployable, single-use devices have been developed for blood collection and DNA separation. Furthermore, a single-use assay card has been developed for performing the real-time PCR reaction with extracted genomic DNA. The assay card integrates DNA extraction, PCR reagent storage and PCR amplification. A prototype field-deployable portable analyzer has also been developed. The analyzer is battery-operated and is capable of assembling the reaction, fluorescence detection and thermal cycling. A proof-of-concept and design feasibility for each of the components of the test has been demonstrated.