Armored long RNA controls or standards for branched DNA assay for detection of human immunodeficiency virus type 1.
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 RNase-resistant 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 degrees 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.
Zhan
Sien
S
Peking Union Medical College, Beijing, People's Republic of China.
Li
Jinming
J
Xu
Ruihuan
R
Wang
Lunan
L
Zhang
Kuo
K
Zhang
Rui
R
D021121:Branched DNA Signal Amplification Assay / Q000379:methods* / Q000592:standards*; D003851:Deoxyribonucleases / Q000378:metabolism; D015658:HIV Infections / Q000175:diagnosis*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D017909:Levivirus / Q000235:genetics; D010957:Plasmids; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D012015:Reference Standards; D012260:Ribonucleases / Q000378:metabolism; D014158:Transcription, Genetic
D012367:RNA, Viral; D003851:Deoxyribonucleases; D012260:Ribonucleases