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.
Subject(s)
HIV Infections/diagnosis , HIV-1/genetics , RNA, Viral/genetics , Real-Time Polymerase Chain Reaction/methods , Viral Load , HIV Infections/blood , HIV-1/metabolism , Humans , RNA, Viral/blood , Real-Time Polymerase Chain Reaction/standards , Reference Standards , Research Design/standards , Sensitivity and SpecificityABSTRACT
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. In addition, R2 value was 0.998, and the slope of the standard curve was -3.33. Conclusion: 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.
Subject(s)
HIV-1/enzymology , RNA, Viral/biosynthesis , Ribonucleases/biosynthesis , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Plasmids/biosynthesis , Plasmids/genetics , Plasmids/pharmacology , RNA, Viral/genetics , Ribonucleases/genetics , Ribonucleases/pharmacologyABSTRACT
BACKGROUND: Remarkable disagreement among different systems of 25-hydroxy vitamin D 25(OH)D assay makes decision making for both clinical and community interventions very difficult. This study aimed to harmonize the results obtained from different 25(OH)D assay systems. METHODS: A total of 275 serum samples were analyzed for 25(OH)D using DIAsource-enzyme immunoassay (EIA), DIAsource-radioimmunoassay (RIA), Roche-electrochemiluminescence (ECL), Diasorin-chemiluminescent immunoassay (CLIA), and high-performance liquid chromatography (HPLC), as the reference method. Serum intact parathyroid hormone (iPTH) was also measured in all samples. Between-system agreement and harmonization were evaluated using Bland-Altman analysis, receiver operating characteristic (ROC), and regression analysis. RESULTS: Mean serum 25(OH)D concentrations and frequency distribution of vitamin D status showed a significant difference among the studied systems (P<.001 for both). Serum 25(OH)D assay results from all systems correlated with those from HPLC. As compared with HPLC, ECL showed a positive bias (+3.8 nmol/L), whereas CLIA had a negative bias (-11.9 nmol/L). Both EIA and RIA showed a more or less similar positive bias (8.0 and 8.1 nmol/L, respectively). Using serum iPTH-based 25(OH)D cutoff points, only ECL results became comparable to and without significant difference with HPLC. However, when system-specific cutoffs were defined based on HPLC results using regression equations, mean 25(OH)D and frequency distribution of vitamin D status were more harmonized compared with the other methods. CONCLUSION: Our findings showed that with adjustment of circulating 25(OH)D based on HPLC, frequency distribution of vitamin D status, as judged by different methods, can be well harmonized with no statistically significant inter-system difference.
