Design and development of a quantitative real time PCR assay for monitoring of HTLV-1 provirus in whole blood.
BACKGROUND
Proviral load quantification of human T-lymphotropic virus type 1 (HTLV-1) is an essential marker for disease progression. Therefore, accurate and precise quantification of the virus is important. However, many articles published about detection and quantification of HTLV-1 virus neither reported any databank for the pre-validation of their primer and probe sequences nor stressed on its importance. Consequently, this failure may cause proviral load measurement variations of different HTLV-1 strains.


OBJECTIVE
The aim of this study was to develop a TaqMan assay for HTLV-1 proviral load quantification which is based on a conserved region of tax gene with minimal sequence variability.


METHODS
For the purpose of finding the most conserved region of tax gene, all the HTLV-1 Gene Bank records including tax gene sequence (524 records by December 2009) were aligned in order to design on the most conserved region of this gene. The specificity, sensitivity, inter and intra assay and the dynamic range of the assay were experimentally determined by their respective methodology.


RESULTS
The assay has a dynamic range of 10-10(7) HTLV-1 plasmid DNA/rxn (reaction) and the limit of detection (LOD) less than 10 copies/rxn. The assay gave coefficient of variation (CV) for the Ct values of less than 1% and 4.8% for intra and inter assay, respectively. Clinical sensitivity and specificity were determined to be 97.8% and 100%, respectively.


CONCLUSIONS
This TaqMan assay is able to reliably quantify proviral load due to the fact that it has been designed on a conserved region of HTLV-1 tax gene with minimal sequence variability.
Department of Biotechnology, Tarbiat Modares University, Tehran, Iran.
Mahmood
M
Naderi
Mahdi
M
Paryan
Kayhan
K
Azadmanesh
Houshang
H
Rafatpanah
Houri
H
Rezvan
Siamak
S
Mirab Samiee
D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D016356:Gene Products, tax / Q000235:genetics*; D015490:HTLV-I Infections / Q000097:blood / Q000821:virology*; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000235:genetics / Q000502:physiology*
D017931:DNA Primers; D004279:DNA, Viral; D016356:Gene Products, tax; C514970:tax protein, Human T-lymphotrophic virus 1