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.

A new label technology for the detection of specific polymerase chain reaction products in a closed tube

“…An estimation of this detection limit can be obtained by extrapolating a standard curve to the point where C t = 0. With our detection technology this detection limit is~10 12 synthesized target amplicons, which is very similar to results obtained with other techniques (22,23). It is possible that this detection limit can be further improved by increasing the labeling degree of the probe and/or by optimizing the position of the label moiety in the probe.…”

“…the results cannot be read directly from the fluorescence counts at different wavelengths. However, even with the best software it is sometimes impossible to distinguish the emissions of two labels from one another, which may result in false positive results (23). A better approach would be the use of fluorescent labels with long excited state lifetimes (such as stable, fluorescent lanthanide chelates) and time-resolved fluorimetry (21) together with prompt labels.…”

Multiplex Reverse Transcriptase-PCR for Simultaneous Detection of Hepatitis B, C, and E Virus

“…16 The majority of these real-time systems either use DNA binding fluorophores such as SYBER Green I or they use specific fluorescently labeled oligonucleotide probes to detect amplification. [16][17][18][19] However, it is also hard to implement the real time detection routinely in majority of tertiary care hospitals in India as well as in other developing countries due to the unavailability of expensive real-time PCR machine, lack of well trained manpower, and the use of expensive fluorophores I or specific fluorescently labeled oligonucleotide required for real time PCR reaction. Therefore, we have developed a sensitive, specific, and costeffective multiplex PCR assay by end point PCR for detection of multiple infectious agents.…”

N-Methyl-d-Aspartic Acid Receptor Expression in the Dorsolateral Prefrontal Cortex of Elderly Patients With Schizophrenia

“…Molecular beacons were used as fluorogenic probes in the real-time PCR. Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to their target nucleic acids (80)(81)(82)(83). The molecular beacons were designed by using a DNA folding program (available at http://www.ibc.wustl.edu/∼zuker) to estimate the stability of the hairpin stem.…”