کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
6135041 | 1593494 | 2010 | 5 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
Quantitation of HTLV-I proviral load by a real-time PCR assay using SYBR Green: Comparison of two methods for DNA isolation
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کلمات کلیدی
موضوعات مرتبط
علوم زیستی و بیوفناوری
ایمنی شناسی و میکروب شناسی
ویروس شناسی
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چکیده انگلیسی
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/106 PBMCs for both methods, with a broad range of quantitation (2.6 log10 to >5 log10), 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.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Virological Methods - Volume 170, Issues 1â2, December 2010, Pages 160-164
Journal: Journal of Virological Methods - Volume 170, Issues 1â2, December 2010, Pages 160-164
نویسندگان
Natalia Andrea Altamirano, Carlos Rocco, Paula Aulicino, Luisa Sen, Andrea Mangano,