Brussel Audrey, Delelis Olivier, Sonigo Pierre
Département des Maladies Infectieuses, Institut Cochin, INSERM U567, CNRS UMR8104, Université René Descartes, Paris, France.
Methods Mol Biol. 2005;304:139-54. doi: 10.1385/1-59259-907-9:139.
An improved Alu-long terminal repeat (LTR) polymerase chain reaction (PCR) assay is described for the quantification of integrated HIV-1 DNA in infected cells. The method includes generation of an infected cell line containing numerous randomly distributed HIV-1 integrated DNA for the construction of the DNA standard and a two-step real-time PCR assay in which the first-round PCR amplifies the DNA sequence between the HIV-1 LTR and the nearest chromosomal Alu element, and the nested PCR specifically amplifies PCR products from the first-round PCR. This assay allows us to quantify proviral DNA with both accuracy and high sensitivity (six proviruses within 50,000 cell equivalents) and exhibits a broad range of quantification spanning 5 log10 provirus copies. This Alu-LTR-based real-time nested PCR assay may be particularly useful to quantify integrated HIV-1 DNA in patients. It may also allow for the precise study of integration of HIV-1 DNA or HIV-1 based lentiviral vectors and may be a valuable tool to test future inhibitors of integration.
描述了一种改进的Alu-长末端重复序列（LTR）聚合酶链反应（PCR）检测方法，用于定量感染细胞中整合的HIV-1 DNA。该方法包括生成一个含有大量随机分布的HIV-1整合DNA的感染细胞系，用于构建DNA标准品，以及两步实时PCR检测，其中第一轮PCR扩增HIV-1 LTR与最接近的染色体Alu元件之间的DNA序列，巢式PCR特异性扩增第一轮PCR的产物。该检测方法使我们能够准确且高灵敏度地定量前病毒DNA（50,000个细胞当量内有六个前病毒），并显示出跨越5个log10前病毒拷贝的广泛定量范围。这种基于Alu-LTR的实时巢式PCR检测方法对于定量患者体内整合的HIV-1 DNA可能特别有用。它还可能允许对HIV-1 DNA或基于HIV-1的慢病毒载体的整合进行精确研究，并且可能是测试未来整合抑制剂的有价值工具。
