Laboratory Branch, Division of HIV/AIDS Prevention, National Center for HIV, STD, and TB Prevention, Centers for Disease Control and Prevention, Atlanta, GA 30333, United States.
J Virol Methods. 2010 Mar;164(1-2):55-62. doi: 10.1016/j.jviromet.2009.11.027. Epub 2009 Dec 3.
In this study, a rapid real-time PCR assay to detect HIV-1 proviral DNA in whole blood was developed using a novel double-stranded primer that does not require a target-specific fluorescent probe or intercalating dye systems. Co-amplification of a human gene RNase P served as the internal control to monitor the efficiency of the DNA extraction and PCR amplification. The HIV-1 amplification efficiency was 100% and could amplify 1 copy of HIV-1 DNA 64% of the time and all attempts to amplify 4 copies were successful in less than 51 min. All 22 HIV-1 sero-positive and 20 sero-negative whole blood specimens tested were classified correctly by this assay. In addition, 22 cultured PBMC specimens infected with various HIV-1 subtypes or CRF (A=2, AC=1, B=4, C=3, D=3, AE=2, F=1, BF=2, G=4) were amplified equally well with a similar threshold cycle (C(t)) number (22.9+/-1.2). The high amplification efficiency and short PCR cycles were in part due to the short target sequence amplified by eliminating the probe-binding sequence between the primers. This assay may be useful as an alternative confirmation test in a variety of HIV testing venues.
本研究开发了一种使用新型双链引物的快速实时 PCR 检测方法，用于检测全血中的 HIV-1 前病毒 DNA。该引物不需要目标特异性荧光探针或嵌入染料系统。人基因 RNase P 的共扩增用作内部对照，以监测 DNA 提取和 PCR 扩增的效率。HIV-1 的扩增效率为 100%，可在 64%的时间内扩增 1 个拷贝的 HIV-1 DNA，所有尝试扩增 4 个拷贝的实验均在 51 分钟内成功。该检测方法可正确分类 22 份 HIV-1 血清阳性和 20 份血清阴性的全血标本。此外，该检测方法还可同等程度地扩增 22 份感染各种 HIV-1 亚型或 CRF（A=2、AC=1、B=4、C=3、D=3、AE=2、F=1、BF=2、G=4）的培养 PBMC 标本，其阈值循环（C(t)）数相似（22.9+/-1.2）。高扩增效率和短 PCR 循环部分归因于通过消除引物之间的探针结合序列来扩增短靶序列。该检测方法可作为各种 HIV 检测场所的替代确认检测方法。
