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

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Ghasemzadegan H.; SHAHABI M.; REZAIE N.; SHARIFI Z.

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Journal: The Scientific Journal of Iranian Blood Transfusion Year:2019 | Volume:16 | Issue:3 Page(s): 194-200

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Title

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

Author(s)

Ghasemzadegan H. | SHAHABI M. | REZAIE N. | SHARIFI Z. | Issue Writer Certificate

Keywords

HTLV-1Q1

ProvirusQ1

Real-Time PCRQ1

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.

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APA: Copy

Ghasemzadegan, H., SHAHABI, M., REZAIE, N., & SHARIFI, Z.. (2019). Design a Real Time PCR with SYBR Green for quantification of HTLV-1 proviral load for blood donors. THE SCIENTIFIC JOURNAL OF IRANIAN BLOOD TRANSFUSION ORGANIZATION (KHOON), 16(3 ), 194-200. SID. https://sid.ir/paper/363102/en

Vancouver: Copy

Ghasemzadegan H., SHAHABI M., REZAIE N., SHARIFI Z.. Design a Real Time PCR with SYBR Green for quantification of HTLV-1 proviral load for blood donors. THE SCIENTIFIC JOURNAL OF IRANIAN BLOOD TRANSFUSION ORGANIZATION (KHOON)[Internet]. 2019;16(3 ):194-200. Available from: https://sid.ir/paper/363102/en

IEEE: Copy

H. Ghasemzadegan, M. SHAHABI, N. REZAIE, and Z. SHARIFI, “Design a Real Time PCR with SYBR Green for quantification of HTLV-1 proviral load for blood donors,” THE SCIENTIFIC JOURNAL OF IRANIAN BLOOD TRANSFUSION ORGANIZATION (KHOON), vol. 16, no. 3 , pp. 194–200, 2019, [Online]. Available: https://sid.ir/paper/363102/en

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