Preparation and Evaluation of Ribonuclease-Resistant Viral HIV RNA Standards Based on Armored RNA Technology

• Published in: Iranian biomedical journal 2018-11, Vol.22 (6), p.394-400
• Main Authors: Gholami, Mohammad, Ravanshad, Mehrdad, Baesi, Kazem, Samiee, Siamak M., Hosseini Rozbahani, Negin, Mohraz, Minoo
• Format: Article
• Language: English
• Subjects: Escherichia coli - genetics; Escherichia coli - metabolism; Full Length; HIV-1 - enzymology; Humans; Plasmids - biosynthesis; Plasmids - genetics; Plasmids - pharmacology; Ribonucleases - biosynthesis; Ribonucleases - genetics; Ribonucleases - pharmacology; RNA, Viral - biosynthesis; RNA, Viral - genetics
• ISSN: 1028-852X; 2008-823X
• DOI: 10.29252/.22.6.394

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. 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. 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. In addition, R2 value was 0.998, and the slope of the standard curve was -3.33. 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.