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.
The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3. Our assay uses tax type-specific primers and dually labeled probes and has a dynamic range of 10(5) to 10 HTLV copies. One hundred sixty-three samples were analyzed, among which all of the different subtypes within each HTLV genotype could be detected. The performance of proviral load determination of our multiplex assay was compared with that of a previously published HTLV-1 singleplex quantitative PCR based on SYBR green detection, developed at a different institute. Linear regression analysis showed a statistically significant (P < 0.0001) and strong (r(2) = 0.87) correlation between proviral load values measured with the two distinct real-time PCR assays. In conclusion, our novel assay offers an accurate molecular diagnosis and genotyping, together with the determination of the proviral load of HTLV-infected individuals, in a single amplification reaction. Moreover, our molecular assay could offer an alternative when current available serological assays are insufficient.
Moens
Britta
B
Rega Institute for Medical Research, KU Leuven, Minderbroederstraat 10, 3000 Leuven, Belgium. britta.moens@rega.kuleuven.be
López
Giovanni
G
Adaui
Vanessa
V
González
Elsa
E
Kerremans
Lien
L
Clark
Daniel
D
Verdonck
Kristien
K
Gotuzzo
Eduardo
E
Vanham
Guido
G
Cassar
Olivier
O
Gessain
Antoine
A
Vandamme
Anne-Mieke
AM
Van Dooren
Sonia
S
D002460:Cell Line; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics; D006800:Deltaretrovirus Infections / Q000175:diagnosis / Q000473:pathology / Q000821:virology*; D016355:Genes, pX / Q000235:genetics; D005838:Genotype; D015368:Human T-lymphotropic virus 1 / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D015367:Human T-lymphotropic virus 2 / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D056187:Human T-lymphotropic virus 3 / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D015203:Reproducibility of Results; D013194:Staining and Labeling / Q000379:methods; D019562:Viral Load
D017931:DNA Primers