Multiplex detection of four pathogenic retroviruses using molecular beacons

We describe a multiplex nucleic acid assay that identifies and determines t he abundance of four different patho genic retroviruses (HIV-1, HIV-2, and human T-lymphotrophic virus types I and II). Retroviral DNA sequences are a mplified in a single, sealed tube by simultaneous PCR assays, and the resul ting amplicons are detected in real time by the hybridization of four diffe rently colored, amplicon-specific molecular beacons. The color of the fluor escence generated in the course of amplification identifies which retroviru ses are present, and the number of thermal cycles required for the intensit y of each color to rise significantly above background provides an accurate measure of the number of copies of each retroviral sequence that were pres ent originally in the sample. Fewer than 10 retroviral genomes can be detec ted. Moreover, 10 copies of a rare retrovirus can be detected in the presen ce of 100,000 copies of an abundant retrovirus. Ninety-six samples can be a nalyzed in 3 hr on a single plate, and the use of a closed-tube format elim inates crossover contamination. Utilizing previously well characterized cli nical samples, we demonstrate that each of the pathogenic retroviruses can be identified correctly and no false positives occur. This assay enables th e rapid and reliable screening of donated blood and transplantable tissues.