Quantitation of HTLV-I proviral load by a real-time PCR assay using SYBR Green: comparison of two methods for DNA isolation.
A real-time quantitative PCR (qPCR) assay using SYBR Green was developed to determine HTLV-I proviral load (pVL) in peripheral blood mononuclear cells (PBMCs), and its performance was evaluated with samples processed as cell lysates and DNA isolated by salting out. Primers targeting the pol region were standardized against the MT2 cell line and HTLV-I copy number was normalized to the amount of cellular DNA by quantitation of the albumin gene. The sensitivity, specificity and reproducibility of the qPCR were assessed in the two methods used for DNA processing. The assay had a limit of detection of 400 HTLV-I copies/10(6) PBMCs for both methods, with a broad range of quantitation (2.6log(10) to >5log(10)), and without cross-reactivity with HTLV-II or with HIV-1. The inter- and intra-assay coefficients of variation were less than 2.4%. HTLV-I pVL quantitation in seven blood donor samples processed as either cell lysates or isolated DNA by salting out showed a strong linear correlation and no difference in the calculated pVL (Fisher's exact test, p>0.05). The assay was found to be a low cost, robust and reproducible assay for quantifying HTLV-I pVL in samples processed as cell lysates or as isolated DNA.
Altamirano
Natalia Andrea
NA
Laboratorio de Biología Celular y Retrovirus, CONICET, Hospital de Pediatría Dr. Juan P. Garrahan, Combate de los Pozos 1881, Cuidad Autónoma de Buenos Aires, Argentina.
Rocco
Carlos
C
Aulicino
Paula
P
Sen
Luisa
L
Mangano
Andrea
A
D052160:Benzothiazoles; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D003959:Diamines; D015751:Genes, pol; D015368:Human T-lymphotropic virus 1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D011804:Quinolines; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019562:Viral Load
D052160:Benzothiazoles; D017931:DNA Primers; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I