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.

FIG. 1.

Sensitivity and specificity of the one-tube, multiplex, real-time PCR assay for the simultaneous detection, differentiation, and quantification of HTLV-1, -2, and -3. (A) HTLV-1, 10-fold dilutions, 10⁵ to 10⁰ copies per PCR (with standard curve); (B) HTLV-2, 10-fold dilutions, 10⁵ to 10⁰ copies per PCR (with standard curve); (C) HTLV-3, 10-fold dilutions, 10⁵ to 10⁰ copies per PCR (with standard curve); and (D) albumin, 10-fold dilutions, 10⁴ to 10⁰ cells per PCR (with standard curve).