One-Step, Multiplex, Real-Time PCR Assay with Molecular Beacon Probes for Simultaneous Detection, Differentiation, and Quantification of Human T-Cell Leukemia Virus Types 1, 2, and 3

Abstract

A single-tube, multiplex, real-time PCR assay with molecular beacons was established in which various probes were used for the simultaneous detection, differentiation, and quantification of human T-cell leukemia virus types 1, 2, and 3 (HTLV-1, HTLV-2, and HTLV-3, respectively) and of simian T-cell leukemia virus types 1 and 3 (STLV-1 and STLV-3, respectively). The quantitative amplification of the standards with MT4 (HTLV-1) and C19 (HTLV-2) cell lines and a molecular clone of HTLV-3 was linear, with the simplex and multiplex methods having similar efficiencies. A maximum difference of 0.9 (mean, 0.4; range, 0.0 to 0.9) was found between threshold cycle values in single and multiplex reactions. The efficiency with each probe in the multiplex reaction was close to 100%, indicating strong linear amplification. The albumin gene was used to standardize the copy number. Comparable results for the detection and quantification of HTLV-1 were obtained with our new methods and with other real-time PCR methods described previously. With our new multiplex assay, however, we were able to detect and quantify HTLV-2 and -3 and STLV-1 and -3 in clinical specimens, with an excellent dynamic range of 10 6 to 10 0 copies per assay, which the other assays could not do. Thus, it will be possible to determine a wide range of HTLV types in both standard and clinical samples, with a detection of 1 to 10 HTLV copies in samples containing at least 100 cells. Furthermore, our system can provide evidence for multiple infections with the three HTLV types, with separate proviral load results. Our new method also could be used for epidemiological studies in Africa and in countries where HTLVs and STLVs are endemic.

Multiplex real-time PCR for the detection and quantitation of HTLV-1 and HTLV-2 proviral load: Addressing the issue of indeterminate HTLV results

“…However, the use of molecular beacons, which are considered more stable, may have reduced or eliminated the need for the inclusion of ROX. 17 In addition, HTLV-3, thus far, has been detected only in Cameroon. 18,19 …”

Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination

“…Multiplex qPCR allows the simultaneous detection and amplification of two or more target DNA sequences in only one amplification reaction. To our knowledge, one specific and one generic biplex qPCR for HTLV-1 and -2 (13,26) and, just recently, one triplex qPCR for HTLV-1, -2, and -3 have been described (3). To address the current problems with HTLV diagnosis and quantitation, taking into account the diversity in HTLV genotypes and subtypes, we developed a novel triplex qPCR assay for simultaneous detection, genotyping, and quantification of PVL of HTLV-1, -2, and -3 infections.…”

A New and Frequent Human T-Cell Leukemia Virus Indeterminate Western Blot Pattern: Epidemiological Determinants and PCR Results in Central African Inhabitants

“…Confirmatory tests are immunofluorescence (IF) (23) but mostly Western blot (WB) analyses (30,35,38,39,64,71). Moreover, research of integrated provirus, in the DNA from peripheral blood cells, could be done by qualitative and/or quantitative PCR (2,4,8,68). Despite some improvements in the WB assays specificity during the last two decades, indeterminate serological patterns are frequent following WB analysis and represent a major concern for routine screening in blood banks in Europe, the Americas, and Africa (7,10,11,16,20,40,61,63).…”