Armored Long RNA Controls or Standards for Branched DNA Assay for Detection of Human Immunodeficiency Virus Type 1

Abstract

The branched DNA (bDNA) assay is a reliable method for quantifying the RNA of human immunodeficiency virus type 1 (HIV-1). The positive controls and standards for this assay for the detection of HIV-1 consist of naked RNA, which is susceptible to degradation by RNase. Armored RNA is a good candidate for an RNaseresistant positive control or standard. However, its use has been limited by the maximal length of the exogenous RNA packaged into virus-like particles by routine armored RNA technology. In the present study, we produced armored long RNA (armored L-RNA) controls or standards (AR-HIV-pol-3034b) for a bDNA assay of HIV-1 by increasing the amount and affinity of the pac sites (the pac site is a specific 19-nucleotide stem-loop region located at the 5 terminus of the MS2 bacteriophage replicase gene) by a one-plasmid double-expression system. AR-HIV-pol-3034b was completely resistant to DNase and RNase, was stable in normal human EDTA-preserved plasma at 4°C for at least 6 months, and produced reproducible, linear results in the Versant HIV-1 RNA 3.0 assay. In conclusion, AR-HIV-pol-3034b could act as a positive control or standard in a bDNA assay for the detection of HIV-1. In addition, the one-plasmid double-expression system can be used as a better platform than the one-plasmid expression system and the two-plasmid coexpression system for expressing armored L-RNA. Human immunodeficiency virus (HIV) infection representsone of the most serious challenges to global public health, since more than 60 million people have been infected with HIV and 25 million have already died of AIDS worldwide (3,22). Accurate determination of HIV type 1 (HIV-1) RNA levels is important for understanding the natural history of HIV infection, predicting the disease progression to AIDS, and determining the efficacy of antiretroviral therapies for a given patient (1,15,16).The branched DNA (bDNA) assay provides a reliable method for quantifying HIV-1 RNA in human plasma. Its lower limit of quantification is 50 copies of HIV-1 RNA/ml. The bDNA assay directly measures HIV-1 RNA by boosting the reporter signal and thus avoids the errors inherent in the extraction and replication of target sequences. This assay is based on the hybridization of HIV-1 RNA to oligonucleotide probes complementary to the most conserved region (about 2.7 kb) of the HIV-1 pol gene and yields a reproducible quantification of HIV-1 RNA that is not affected by the sequence variability of HIV-1 subtypes (8,12,17,28).The bDNA assay for HIV-1 depends on the use of RNA synthesized by in vitro transcription for its positive controls and standards. A major disadvantage of using naked RNA is that it is susceptible to degradation by RNase. Thus, there is a need for RNase-resistant RNA controls and standards.Armored RNA is a kind of noninfectious recombinant viruslike particle (VLP) containing target exogenous RNA. It is the most suitable candidate for a positive control or standard for the quantification of an RNA virus, because it is RNase resistant, stable, noninfectio...

Failure to detect Xenotropic murine leukaemia virus-related virus in Chinese patients with chronic fatigue syndrome

“…This sequence was obtained by overlapping extension PCR [11,12] amplification of XMRV (nt 33 to 1149, 1117 bp amplified from plasmid VP62 [3], kindly provided by Lombardi; [Genbank: EF185282]) and HCV (nt 25 to 445, 420 bp amplified from pNCCL-HCV [13], constructed by our laboratory; [Genbank: AF139594]). The primers used in this method are shown in Table 1.…”

Design of virus-based nanomaterials for medicine, biotechnology, and energy

“…In the original area of HIV detection, Armored RNA was expanded beyond RT-PCR assays to the branched DNA (bDNA) assay, which provides a reliable method for quantification of HIV-1 RNA but requires a longer 3 kb RNA control. 414 The standard strategy for RNA encapsulation within MS2 is limited to only around 2 kb, but increasing the packaging efficiency to overcome this limit can be achieved through incorporating more translational repressor stem-loops within the RNA, which specifically interact with the MS2 capsid and trigger the self-assembly of the viral shell around the cargo. 415 In such a manner, Armored RNA containing the longer HIV pol gene was successfully synthesized and performed reliably as a standard for the bDNA assay.…”

Preparation of MS2 Phage-Like Particles and Their Use As Potential Process Control Viruses for Detection and Quantification of Enteric RNA Viruses in Different Matrices

“…Moreover, MS2 PLP can be stored for a long time without degradation (WalkerPeach et al, 1999; Beld et al, 2004; Hietala and Crossley, 2006; Wei Y.X. et al, 2008; Yu et al, 2008; Zhan et al, 2009; Song et al, 2011). Another positive feature of MS2 PLP is that they are not infectious and thus working with them is safe for laboratory personnel.…”