A novel sensitive multiplex RT-PCR ELISA method for detection of HIV-1/HCV co-infection

Despite sensitive antibody based blood donor screening, infection can be transmitted during window period. Therefore sensetive methods bosed on nucleic acid tests (NATs) have been considered. The aim of this project was to design a more sensitive method for detection of PCR products and diagnosis of HIV-1/HCV co-infection accurately. After designing specific primers and probes, the Multiplex RT-PCR method was optimized and the PCR products were labeled with Digoxigenin. The PCR product was denatured under alkaline condition and was hybridized with the specific probe that had a biotin at 5' end, and then was added to streptavidin coated wells. After washing an antibody against DIG, conjugated with alkaline phosphates enzyme was used, following second washing, the substrate (ABTS) solution was added to each reaction well. Development of green color shows the positive where as no color shows negative results. 35 samples were tested with the developed method including 27 positive samples, 8 confirmed negative and 4 standard panels. False negative or positive reactions were not observed. This method had acceptable sensitivity and specificity for detecting HCV and HIV-1 infections during window period, also the method can be quantified which can be used for the flow-up and treatment of patients. In addition to the very high sensetivity of the test, it is cost effective and takes less time to performe.