Design and Development of a Quantitative Taqman Real-Time Pcr Assay for Evaluation of Hiv-1 (Group M) Viral Load in Plasma Using Armored Rna Standard

Design and Development of a Quantitative Taqman Real-Time Pcr Assay for Evaluation of Hiv-1 (Group M) Viral Load in Plasma Using Armored Rna Standard Publisher Pubmed

Gholami M¹ ; Baesi K² ; Ravanshad M¹ ; Samiee SM³ ; Rouzbahani NH⁴ ; Mohraz M⁵

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1. Department of Medical Virology, Faculty of Medical Sciences, Tarbiat Modares University, Al Ahmad Street, Tehran, Iran
2. Hepatitis and AIDS Department, Pasture Institute of Iran, Tehran, Iran
3. Food and Drug Laboratory Research Center, Ministry of Health and Medical Education, Tehran, Iran
4. Department of Immunology, Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
5. Iranian Research Center for HIV AIDS (IRCHA), Iranian Institute for Reduction of High-Risk Behaviors Tehran University of Medical Sciences, Keshavarz Blvd, Nosrat St, Tehran, Iran

Source: Clinical Laboratory Published:2018

Abstract

Background: Human immunodeficiency virus-1 (HIV-1) is a viral infectious agent that gradually extinguishes the immune system, resulting in the acquired immune deficiency syndrome (AIDS). The aim of this study was to develop a TaqMan based detection assay to evaluate HIV-1 plasma viral load and to construct a stable internal positive control (IPC) and external positive control (EPC) RNA based on Armored RNA (AR) technology. Methods: The MS2 maturase, coat protein gene and HIV-1 pol gene were cloned in pET-32a plasmid. The recently fabricated recombinant plasmid was transformed into Escherichia coli strain BL2 (DE3) and protein expression and Armored RNA was fabricated in presence of isopropyl-L-Thio-D-galactopyranoside (IPTG). The Armored RNA was precipitated and purified by polyethylene glycol (PEG) and sephacryl S-200 chromatography. The stability of Armored RNA was evaluated by treatment with DNase I and RNase A and confirmed by transmission electron microscopy (TEM) and gel agarose electrophoresis. The specificity, sensitivity, inter-And intra-day precision, and the dynamic range of the assay were experimentally determined. Results: The AR was stable in presence of ribonuclease, and the assay had a dynamic detection range from 101 to 105 copies of AR. The coefficient of variation (CV) was 4.8% for intra-Assay and 5.8% for inter-Assay precision. Clinical specificity and sensitivity of the assay were assessed at 100% and 96.66%, respectively. The linear regression analysis confirmed a high correlation between the in-house and the commercial assay, Real Star HIV-1-qRTPCR, respectively (R2 = 0.888). Conclusions: The AR standard is non-infectious and highly resistant in the presence of ribonuclease. The TaqMan assay developed is able to quantify HIV viral load based on a novel conserved region of HIV-1 pol gene which has minimal sequence inconsistency. © 2018 Verlag Klinisches Labor GmbH. All rights reserved.

2. The Relationship Between Hiv Antibody Titer, Hiv Viral Load, Hiv P24 Antigen, and Cd4 T-Cell Count Among Iranian Hiv-Positive Patients, Infectious Disorders - Drug Targets (2020)

Experts (# of related papers)

Seyed Ahmad Seyed Alinaghi (1)

Style	Citing Format
MLA	Gholami M, et al.. "Design and Development of a Quantitative Taqman Real-Time Pcr Assay for Evaluation of Hiv-1 (Group M) Viral Load in Plasma Using Armored Rna Standard." Clinical Laboratory, vol. 64, no. 6, 2018, pp. 955-963.
APA	Gholami M, Baesi K, Ravanshad M, Samiee SM, Rouzbahani NH, Mohraz M (2018). Design and Development of a Quantitative Taqman Real-Time Pcr Assay for Evaluation of Hiv-1 (Group M) Viral Load in Plasma Using Armored Rna Standard. Clinical Laboratory, 64(6), 955-963.
Chicago	Gholami M, Baesi K, Ravanshad M, Samiee SM, Rouzbahani NH, Mohraz M. "Design and Development of a Quantitative Taqman Real-Time Pcr Assay for Evaluation of Hiv-1 (Group M) Viral Load in Plasma Using Armored Rna Standard." Clinical Laboratory 64, no. 6 (2018): 955-963.
Harvard	Gholami M et al. (2018) 'Design and Development of a Quantitative Taqman Real-Time Pcr Assay for Evaluation of Hiv-1 (Group M) Viral Load in Plasma Using Armored Rna Standard', Clinical Laboratory, 64(6), pp. 955-963.
Vancouver	Gholami M, Baesi K, Ravanshad M, Samiee SM, Rouzbahani NH, Mohraz M. Design and Development of a Quantitative Taqman Real-Time Pcr Assay for Evaluation of Hiv-1 (Group M) Viral Load in Plasma Using Armored Rna Standard. Clinical Laboratory. 2018;64(6):955-963.
BibTex	@article{ author = {Gholami M and Baesi K and Ravanshad M and Samiee SM and Rouzbahani NH and Mohraz M}, title = {Design and Development of a Quantitative Taqman Real-Time Pcr Assay for Evaluation of Hiv-1 (Group M) Viral Load in Plasma Using Armored Rna Standard}, journal = {Clinical Laboratory}, volume = {64}, number = {6}, pages = {955-963}, year = {2018} }
RIS	TY - JOUR AU - Gholami M AU - Baesi K AU - Ravanshad M AU - Samiee SM AU - Rouzbahani NH AU - Mohraz M TI - Design and Development of a Quantitative Taqman Real-Time Pcr Assay for Evaluation of Hiv-1 (Group M) Viral Load in Plasma Using Armored Rna Standard JO - Clinical Laboratory VL - 64 IS - 6 SP - 955 EP - 963 PY - 2018 ER -

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