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Journal of Clinical Microbiology, April 2009, p. 1129-1135, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.02006-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Guillaume Besson1 and Mirdad Kazanji1,2*

Unité de Rétrovirologie, Centre International de Recherches Médicales de Franceville (CIRMF), BP 769, Franceville, Gabon,1 Réseau International des Instituts Pasteur, Institut Pasteur, 28 Rue du Dr Roux, 75015 Paris, France2

Received 17 October 2008/ Returned for modification 22 December 2008/ Accepted 3 February 2009

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 106 to 100 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.

* Corresponding author. Mailing address: Centre International de Recherche Médicale de Franceville, BP 769, Franceville, Gabon. Phone: 241 07 69 90 99. Fax: 241 67 72 95. E-mail: m.kazanji{at}cirmf.org

{triangledown} Published ahead of print on 11 February 2009.

Journal of Clinical Microbiology, April 2009, p. 1129-1135, Vol. 47, No. 4
0095-1137/09/$08.00+0     doi:10.1128/JCM.02006-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Moens, B., Lopez, G., Adaui, V., Gonzalez, E., Kerremans, L., Clark, D., Verdonck, K., Gotuzzo, E., Vanham, G., Cassar, O., Gessain, A., Vandamme, A.-M., Van Dooren, S. (2009). 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. J. Clin. Microbiol. 47: 3682-3691 [Abstract] [Full Text]  


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