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Preparation and Evaluation of Ribonuclease-Resistant Viral HIV RNA Standards Based on Armored RNA Technology
Mohammad Gholami , Mehrdad Ravanshad , Kazem Baesi , Siamak M. Samiee , Negin Hosseini Rozbahani , Minoo Mohraz
Abstract:  
Background: The human immunodeficiency virus type 1 (HIV-1) is an infectious viral agent that gradually extinguishes the immune system, resulting in acquired immune deficiency syndrome (AIDS). The aim of this study was to construct an RNA-positive control based on armored (AR) RNA technology, using HIV-1 RNA as a model. Methods: The MS2 maturase, a coat protein gene (at positions 1765 to 1787) and HIV-1 pol gene were cloned into pET-32a plasmid. The prepared plasmid was transformed into Escherichia coli strain BL2 (DE3), and the expression of the construct was induced by 1 mM of isopropyl-L-thio-D-galactopyranoside (IPTG) at 37 °C for 16 h to obtain the fabricated AR RNA. The AR RNA was precipitated and purified using polyethylene glycol and Sephacryl S-200 chromatography. Results: The stability of AR RNA was evaluated by treatment with DNase I and RNase A and confirmed by transmission electron microscopy and gel agarose electrophoresis. Tenfold serial dilution of AR RNA from 101 to 105 was prepared; real-time PCR assays had a range of detection between 101 and 105, R2 value was 0.998, and the slope of the standard curve was -3.33. Conclusions: Prepared AR RNA, as a positive control, could be used as a basis for launching an in-house HIV-1 virus assay and other infectious agents. It can be readily available to laboratories and HIV research centers. The AR RNA is non-infectious and highly resistant to ribonuclease enzyme and can reduce the risk of infection in the clinical laboratory.
Keywords: HIV-1, Real-time PCR, Virus-like particle
Full-Text [PDF 809 kb]      
Type of Study: Full Length | Subject: Molecular Microbiology
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