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Journal of Clinical Microbiology, August 2009, p. 2571-2576, Vol. 47, No. 8
0095-1137/09/$08.00+0     doi:10.1128/JCM.00232-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Sien Zhan,1,2 Jinming Li,2* Ruihuan Xu,1,2 Lunan Wang,2 Kuo Zhang,2 and Rui Zhang2

Graduate School, Peking Union Medical College, Chinese Academy of Medical Sciences,1 National Center for Clinical Laboratories, Beijing Hospital, Beijing, People's Republic of China2

Received 4 February 2009/ Returned for modification 23 March 2009/ Accepted 28 May 2009

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°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.

* Corresponding author. Mailing address: National Center for Clinical Laboratories, 1 Dahua Road, Dongdan, Beijing 100730, People's Republic of China. Phone: 86-10-58115053. Fax: 86-10-65212064. E-mail: ljm63hn{at}yahoo.com.cn

{triangledown} Published ahead of print on 3 June 2009.

Journal of Clinical Microbiology, August 2009, p. 2571-2576, Vol. 47, No. 8
0095-1137/09/$08.00+0     doi:10.1128/JCM.00232-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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