Armored Long RNA Controls or Standards for Branched DNA Assay for Detection of Human Immunodeficiency Virus Type 1
Armored Long RNA Controls or Standards for Branched DNA Assay for Detection of Human Immunodeficiency Virus Type 1
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用于检测 1 型人类免疫缺陷病毒的分支 DNA 测定的装甲长 RNA 对照或标准品
DOI：
10.1128/jcm.00232-09
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发表时间：
2009-08-01
影响因子：
9.4
通讯作者：
Zhang, Rui
中科院分区：
文献类型：
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作者：
Zhan, Sien;Li, Jinming;Zhang, Rui
分支DNA（bDNA）检测是定量人类免疫缺陷病毒1型（HIV-1）RNA的可靠方法。用于检测HIV-1的本试验的阳性对照品和标准品由裸RNA组成，其易被RNA酶降解。装甲RNA是RNase抗性阳性对照或标准品的良好候选物。然而，其使用受到常规装甲RNA技术包装成病毒样颗粒的外源RNA的最大长度的限制。在本研究中，我们通过单质粒双表达系统增加pac位点（pac位点是位于MS 2噬菌体复制酶基因5'末端的特异性19个核苷酸的茎环区域）的数量和亲和力，制备了用于HIV-1 bDNA检测的装甲长RNA（装甲L-RNA）对照或标准品（AR-HIV-pol-3034 b）。AR-HIV-pol-3034 b对DNA酶和RNA酶完全耐药，在正常人EDTA保存血浆中于4 ℃下稳定至少6个月，并在Versant HIV-1 RNA 3.0检测中产生可重现的线性结果。总之，AR-HIV-pol-3034 b可作为bDNA检测试剂盒中的阳性对照品或标准品，用于检测HIV-1。此外，单质粒双表达系统可以作为比单质粒表达系统和双质粒共表达系统更好的表达装甲L-RNA的平台。
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.