Multiplex detection of four pathogenic retroviruses using molecular beacons

Abstract

We describe a multiplex nucleic acid assay that identifies and determines the abundance of four different pathogenic retroviruses (HIV-1, HIV-2, and human T-lymphotrophic virus types I and II). Retroviral DNA sequences are amplified in a single, sealed tube by simultaneous PCR assays, and the resulting amplicons are detected in real time by the hybridization of four differently colored, amplicon-specific molecular beacons. The color of the fluorescence generated in the course of amplification identifies which retroviruses are present, and the number of thermal cycles required for the intensity of each color to rise significantly above background provides an accurate measure of the number of copies of each retroviral sequence that were present originally in the sample. Fewer than 10 retroviral genomes can be detected. Moreover, 10 copies of a rare retrovirus can be detected in the presence of 100, 000 copies of an abundant retrovirus. Ninety-six samples can be analyzed in 3 hr on a single plate, and the use of a closed-tube format eliminates crossover contamination. Utilizing previously well characterized clinical samples, we demonstrate that each of the pathogenic retroviruses can be identified correctly and no false positives occur. This assay enables the rapid and reliable screening of donated blood and transplantable tissues.

Figure 1

Principal of operation of molecular beacons. Free molecular beacons are nonfluorescent because the hairpin stem keeps the fluorophore close to the quencher. When the probe sequence in the hairpin loop hybridizes to its target, forming a rigid double helix, a conformational change occurs that removes the quencher from the vicinity of the fluorophore, thereby restoring fluorescence.

Figure 2

Real-time detection of four different retroviral DNAs in a multiplex format. Four assays were carried out in sealed tubes, each initiated with 100,000 molecules of a different retroviral DNA. Each reaction contained four sets of PCR primers specific for unique HIV-1, HIV-2, HTLV-I, and HTLV-II nucleotide sequences and four molecular beacons, each specific for one of the four amplicons and labeled with a differently colored fluorophore. Fluorescence from the fluorescein-labeled molecular beacon (HIV-1-specific) is plotted in red, fluorescence from the tetrachlorofluorescein-labeled molecular beacon (HIV-2-specific) is plotted in green, fluorescence from the tetramethylrhodamine-labeled molecular beacon (HTLV-I-specific) is plotted in blue, and fluorescence from the rhodamine-labeled molecular beacon (HTLV-II-specific) is plotted in brown. The slight HTLV-I signal seen in the assay initiated with HTLV-II DNA is an artifact that resulted from a portion of the rhodamine fluorescence being interpreted by the spectrofluorometric thermal cycler as tetramethylrhodamine fluorescence.

Figure 3

Detection of a rare retroviral target in the presence of an abundant retroviral target. Five multiplex assays were initiated with 10⁵ molecules of HTLV-I DNA and either 10⁵, 10⁴, 10³, 10², or 10¹ molecules of HIV-2 DNA, and a sixth multiplex assay, which served as a control, did not contain any template DNA. Each assay contained four sets of PCR primers and four differently colored molecular beacons. (Upper) Fluorescence from the tetramethylrhodamine-labeled molecular beacons, which is due to the synthesis of HTLV-I amplicons. (Lower) Fluorescence from the tetrachlorofluorescein-labeled molecular beacons, which is due to the synthesis of HIV-2 amplicons. The number of molecules of each retroviral DNA that were present originally in each assay tube is indicated to the right of each curve.

Figure 4

Inverse linear relationship between the number of thermal cycles it takes for enough amplicons to be synthesized for a significant fluorescent signal to appear (threshold cycle) and the logarithm of the number of retroviral target molecules originally present in a sample. The threshold cycle for each fluorophore was reached when the intensity of the fluorescent signal was 10 times as great as the standard deviation of the background fluorescence. The results demonstrate that quantitative determinations can be made over an extremely wide range of target concentrations, and they show that the assay is sufficiently sensitive to detect as little as 10 molecules of retroviral DNA.