Design a Real Time PCR with SYBR Green for quantification of HTLV-1 proviral load for blood donors

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.