Multiplex detection of four pathogenic retroviruses using molecular beacons
Multiplex detection of four pathogenic retroviruses using molecular beacons
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DOI：
10.1073/pnas.96.11.6394
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发表时间：
1999-05-25
影响因子：
11.1
通讯作者：
Kramer, FR
中科院分区：
文献类型：
--
作者：
Vet, JAM;Majithia, AR;Kramer, FR
我们描述了一种多重核酸检测方法，该方法可识别并确定四种不同致病性逆转录病毒（HIV - 1、HIV - 2以及人类T淋巴细胞病毒I型和II型）的数量。逆转录病毒DNA序列在单个密封管中通过同时进行的聚合酶链反应（PCR）检测进行扩增，并且通过四种不同颜色的、扩增产物特异性分子信标杂交对所得扩增产物进行实时检测。扩增过程中产生的荧光颜色可确定存在哪些逆转录病毒，每种颜色的强度显著高于背景所需的热循环次数可准确测量样本中最初存在的每种逆转录病毒序列的拷贝数。可检测到少于10个逆转录病毒基因组。此外，在100,000个大量存在的逆转录病毒拷贝存在的情况下，可检测到10个罕见逆转录病毒的拷贝。在一块板上可在3小时内分析96个样本，并且使用闭管形式可消除交叉污染。利用先前充分表征的临床样本，我们证明每种致病性逆转录病毒都能被正确识别，且无假阳性出现。这种检测方法能够对捐献的血液和可移植组织进行快速且可靠的筛查。
We describe a multiplex nucleic acid assay that identifies and determines the abundance of four different patho genic retroviruses (HIV-1, HIV-2, and human T-lymphotrophic virus types I and II). Retroviral DNA sequences are amplified in a single, sealed tube by simultaneous PCR assays, and the resulting amplicons are detected in real time by the hybridization of four differently colored, amplicon-specific molecular beacons. The color of the fluorescence generated in the course of amplification identifies which retroviruses are present, and the number of thermal cycles required for the intensity of each color to rise significantly above background provides an accurate measure of the number of copies of each retroviral sequence that were present originally in the sample. Fewer than 10 retroviral genomes can be detected. Moreover, 10 copies of a rare retrovirus can be detected in the presence of 100,000 copies of an abundant retrovirus. Ninety-six samples can be analyzed in 3 hr on a single plate, and the use of a closed-tube format eliminates crossover contamination. Utilizing previously well characterized clinical samples, we demonstrate that each of the pathogenic retroviruses can be identified correctly and no false positives occur. This assay enables the rapid and reliable screening of donated blood and transplantable tissues.