:: Volume 16, Issue 3 (Autumn 2019) ::
Sci J Iran Blood Transfus Organ 2019, 16(3): 194-200 Back to browse issues page
Design a Real Time PCR with SYBR Green for quantification of HTLV-1 proviral load for blood donors
H. Ghasemzadegan, M. Shahabi Dr., N. Rezaei Dr., Z. Sharifi Dr.
Abstract:   (563 Views)
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 R2 = 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. 
 
 
Keywords: Key words: HTLV-1, Provirus, Real-Time PCR
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Type of Study: Research | Subject: Virology


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Volume 16, Issue 3 (Autumn 2019) Back to browse issues page