Topographic and quantitative display of integrated human immunodeficiency virus-1 provirus DNA in human lymph nodes by real-time polymerase chain reaction
Topographic and quantitative display of integrated human immunodeficiency virus-1 provirus DNA in human lymph nodes by real-time polymerase chain reaction
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DOI：
10.1016/s1525-1578(10)60548-x
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发表时间：
2005-05-01
影响因子：
4.1
通讯作者：
Schmitz, H
中科院分区：
文献类型：
--
作者：
Drosten, C;M端ller-Kunert, E;Schmitz, H
原位聚合酶链反应（isPCR）已应用于许多领域，需要检测基因组标记结合其在组织中的拓扑定位。我们在这里描述了一种新的方法，该方法规避了原位PCR的主要缺点，即灵敏度低，DNA从细胞中泄漏，以及无法量化DNA输入。将来自人类免疫缺陷病毒（HIV）-1感染患者的淋巴结的冷冻切片固定在玻璃显微镜载玻片上，并使用金刚石切割装置将玻璃刻划成边长为0-5 mm的方形碎片。然后将载玻片附着到粘合剂、弹性塑料箔上，最后将其破碎，并将箔延伸以允许将片段分选到PCR微量滴定板中。通过灵敏的实时PCR方案检测材料的HIV-1前病毒DNA。对随后的切片进行滤泡树突状细胞染色以鉴定滤泡。片段化过程防止扩增的DNA泄漏到邻近区域，如原位PCR经常经历的那样。前病毒明显与滤泡区域相关，其中携带前病毒的细胞平均占总细胞群的0.8%（峰值密度，所有滤泡细胞的3.1%）。这种方法的结果表明，高密度的卵泡中含有前病毒的细胞可能是重要的持久性的前病毒DNA在感染的人。
In situ polymerase chain reaction (isPCR) has been applied in many fields that require detection of a genomic marker in combination with its topographic localization in tissue. We describe here a novel approach that circumvents the major drawbacks of in situ PCR, ie, low sensitivity, leakage of DNA from cells, and inability to quantify the DNA input. Frozen sections of a lymph node from a human immunodeficiency virus (HIV)-1-hifected patient were fixed on glass microscope slides, and the glass was scored into square fragments of 0-5-mm edge length using a diamond cutting device. Slides were then attached to adhesive, elastic plastic foil and finally broken, and the foil was extended to allow sorting of fragments into PCR microtiter plates. The material was tested for HIV-1 proviral DNA by a sensitive real-time PCR protocol. Subjacent sections were stained for follicular dendritic cells to identify follicles. The fragmentation process prevented leakage of amplified DNA to neighboring areas as often experienced with in situ PCR. Provirus was clearly associated with follicular areas, in which provirus-carrying cells represented an average of 0.8% of the total cell population (peak density, 3.1% of all follicular cells). The results of this method suggest that the high density of provirus-containing cells in follicles may be important for the persistence of proviral DNA in infected persons.