序列捕获聚合酶链反应可提高临床标本中分枝杆菌DNA的检测率。
Sequence capture-PCR improves detection of mycobacterial DNA in clinical specimens.
作者信息
Mangiapan G, Vokurka M, Schouls L, Cadranel J, Lecossier D, van Embden J, Hance A J
机构信息
Institut National de la Santé et de la Recherche Médicale U.82, Faculté de Médecine Xavier Bichat, Paris, France.
出版信息
J Clin Microbiol. 1996 May;34(5):1209-15. doi: 10.1128/jcm.34.5.1209-1215.1996.
The rapid identification of mycobacterial DNA in clinical samples by PCR can be useful in the diagnosis of tuberculous infections, but several large studies have found that the sensitivity of this approach is not better than that of culture. In order to improve the sensitivity of detection of mycobacterial DNA in clinical specimens from patients with paucibacillary forms of tuberculosis, we have developed a procedure permitting the specific capture of mycobacterial DNA in crude samples prior to amplification, thereby concentrating the target sequences and removing irrelevant DNA and other potential inhibitors of the amplification reaction (sequence capture-PCR). By using this approach to capture and amplify two different sequences specific for organisms of the Mycobacterium tuberculosis complex (IS6110 and the direct repeat region), it was possible to detect as little as one genome of mycobacterial DNA in samples containing up to 750 micrograms of total DNA, representing a 10- to 100-fold increase in sensitivity compared with that obtained by purifying total DNA prior to amplification. Detection of the IS6110 sequence in pleural fluid samples from patients with tuberculous pleurisy by sequence capture-PCR gave positive results in 13 of 17 cases, including 3 of 3 culture-positive samples and 10 of 14 culture-negative samples. In contrast, when total DNA was purified from these samples by adsorption to a silica matrix prior to amplification, only the three culture-positive samples were positive by PCR. The sensitivity of detection of the direct repeat sequence in these samples by sequence capture-PCR was similar to that of IS6110 and, in addition, permitted immediate typing of the strains from some patients. We conclude that sequence capture-PCR improves the sensitivity of detection of mycobacterial DNA in paucibacillary samples. This approach should be useful in detecting rare target sequences from organisms implicated in other pathologic processes.
通过聚合酶链反应（PCR）快速鉴定临床样本中的分枝杆菌DNA，对结核感染的诊断可能有用，但几项大型研究发现，这种方法的敏感性并不优于培养法。为了提高在少菌型肺结核患者临床标本中检测分枝杆菌DNA的敏感性，我们开发了一种程序，允许在扩增前从粗样本中特异性捕获分枝杆菌DNA，从而浓缩靶序列并去除无关DNA和扩增反应的其他潜在抑制剂（序列捕获-PCR）。通过使用这种方法捕获并扩增结核分枝杆菌复合群生物体特异的两个不同序列（IS6110和直接重复区域），在含有高达750微克总DNA的样本中，有可能检测到低至一个基因组的分枝杆菌DNA，与扩增前纯化总DNA相比，敏感性提高了10至100倍。通过序列捕获-PCR检测结核性胸膜炎患者胸腔积液样本中的IS6110序列，17例中有13例呈阳性结果，包括3例培养阳性样本中的3例和14例培养阴性样本中的10例。相比之下，当在扩增前通过吸附到硅胶基质从这些样本中纯化总DNA时，只有3例培养阳性样本通过PCR呈阳性。通过序列捕获-PCR检测这些样本中直接重复序列的敏感性与IS6110相似，此外，还能对一些患者的菌株立即进行分型。我们得出结论，序列捕获-PCR提高了少菌型样本中分枝杆菌DNA检测的敏感性。这种方法在检测涉及其他病理过程的生物体的罕见靶序列方面应该是有用的。
Zotero 插件