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Development and validation of a Multiplex Real-Time PCR for HTLV-1/2 confirmatory diagnosis

Accurate diagnostic tests are powerful tool to control the spread of the human T-lymphotropic virus (HTLV) infection. In Brazil, the currently applied diagnostic algorithm for HTLV-1/2 screening is based on serological tests (enzyme-linked immunosorbent assay followed by Western Blot). However this algorithm is unsuitable due to its high cost and the elevated rate of Western Blot (WB) indeterminate results. Nevertheless, molecular techniques such as real-time PCR (qPCR) can overlap these drawbacks because of their high sensitivity and specificity. Several studies have described different qPCR protocols for HTLV-1/2 diagnosis but they lack suitable validation. For this reason, we developed and validated a qualitative multiplex qPCR platform for simultaneous detection and discrimination of the HTLV-1/2 infection in a single reaction tube. A total of 73 HTLV-positive samples and 100 samples obtained from non-infected individuals were tested. The detection limit was one copy/reaction for HTLV-1 and 10 copies/reaction for HTLV-2. We observed a high and similar efficiency between the single and multiplex format as well as the cycle treshold and r2 values. In addition, this molecular platform reached 100% of sensitivity and specificity. In conclusion, the developed method using one tube multiplex qPCR (confirmatory and discriminatory) for HTLV-1/2 was validated. It showed low cost and high sensitivity and specificity compared to previously described assays and to the traditional confirmatory method (WB). Therefore, this platform can be a supportive tool for the current confirmatory methods adopted.

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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

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Rocha Júnior, M.C., Haddad, R., de Deus Wagatsuma, V.M. et al. Development and validation of a Multiplex Real-Time PCR for HTLV-1/2 confirmatory diagnosis. Retrovirology 11 (Suppl 1), P105 (2014).

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