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Design a Real Time PCR with SYBR Green for quantification of HTLV-1 proviral load for blood donors
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H. Ghasemzadegan   , M. Shahabi  , N. Rezaei , Z. Sharifi |
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| Keywords: Key words: HTLV-1, Provirus, Real-Time PCR |
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Type of Study: Research |
Subject:
Virology
Published: 2019/10/2
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Full-Text: (1510 Views) |
References:
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Sci J Iran Blood Transfus Organ 2019;16 (3): 194-200
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Design a Real Time PCR with SYBR Green for
quantification of HTLV-1 proviral load for blood donors
Ghasemzadegan H.1, Shahabi M.1, Rezaie N.2, Sharifi Z.1
1Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran
2Institute of Endocrinology and Metabolism Research and Training Center, Tehran University of Medical Sciences, Tehran, Iran
Abstract
Background and Objectives
In Iran, Khorasan province is an endemic area for HTLV-1 virus. Considering the inability of serological tests to determine HTLV-1 in window period, their failure to confirm the indetermination results of western blot, and given the probability for HTLV-1 transfusion transmission, a SYBR green-based Real Time PCR was set to measure the HTLV-1 proviral load.
Materials and Methods
In this experimental study, using a cloning method and drawing a standard curve, the Real -Time PCR test was run to determine the HTLV-1 proviral load. At first, genomic DNA was extracted from peripheral blood mononuclear cells. Then, the PCR product of the Tax gene was placed in a cloning vector and recombinant plasmid was diluted by drawing a standard curve and a real-time PCR test was conducted using SYBR Green method.
Results
Cloning was performed using PCR product for tax gene, pTZ57/T vector, and E. coli (TG1 strain). Cloning accuracy was confirmed with Colony PCR and sequencing and used as the Real-Time PCR test standard. The standard curve was drawn with serial dilutions of recombinant plasmid containing Tax-1 gene. The slope of the standard curve was 3.3 and R 2 = 0.99 which indicates the linearity and efficiency of the test reaction.
Conclusions
Real - Time PCR method is an appropriate method to measure HTLV-1 proviral load.
Key words: HTLV-1, Provirus, Real-Time PCR
Received: 18 Feb 2019
Accepted: 13 Aug 2019
Correspondence: Sharifi Z., PhD of Virology. Professor of Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine.
P.O.Box: 14665-1157, Tehran, Iran. Tel: (+9821) 82052152; Fax: (+9821) 88601555
E-mail: sharifiz@yahoo.com |
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Ghasemzadegan H, Shahabi M, Rezaei N, Sharifi Z. Design a Real Time PCR with SYBR Green for quantification of HTLV-1 proviral load for blood donors. Sci J Iran Blood Transfus Organ 2019; 16 (3) :194-200 URL: http://bloodjournal.ir/article-1-1248-en.html
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