A novel, unique, rapid and reliable multiplex RT-PCR test will be developed for the simultaneous detection of five viral pathogens, HTLV- I/II, HIV-1, HBV and HCV, frequently associated with transfusion- transmitted diseases. Reverse transcription of HCV RNA, and amplification of HTLV-I/ II, HIV-1 and HBV DNAs will be carried out simultaneously in a single reaction tube using a combination of primers specific for these pathogens. The PCR products will be detected in microtiter capture plates designed for their simultaneous or individual detection. Simplified procedures will be evaluated to establish the optimum conditions for rapid collection and processing of whole blood samples, while preserving the integrity of both the RNA and DNA. The transcription and amplification conditions for the simultaneous amplification of all five analytes in a single reaction tube using a multiplex RT-PCR approach will be worked out. The sensitivity, specificity and reproducibility of multiplex RT-PCR and detection assay will be validated on appropriate quality controls and blood samples from infected individuals. In the course of Phase II, these studies will be extended to further refine and evaluate multiplex RT-PCR kits for suitability of use in a clinical laboratory setting.
The multiplex PCR test will provide a highly sensitive, specific and cost effective means for simultaneous testing of infectious agents commonly transmitted through blood products. This test can be used for confirmation of indeterminate results from initial screening of blood donations and will also be useful for screening high risk populations for the early appearance of HTLV-I/II, HIV-1, HBV, or HCV infections.
