Brugière O, Vokurka M, Lecossier D, Mangiapan G, Amrane A, Milleron B, Mayaud C, Cadranel J, Hance A J
INSERM U.82, Faculté de Médecine Xavier Bichat, Centre de Pneumologieet de Réanimation Respiratoire, Paris, France.
Am J Respir Crit Care Med. 1997 Apr;155(4):1478-81. doi: 10.1164/ajrccm.155.4.9105098.
Techniques based on the polymerase chain reaction (PCR) can be used to rapidly identify DNA from Mycobacterium tuberculosis in clinical samples from patients with tuberculosis, but prior studies evaluating this approach in the diagnosis of paucibacillary forms of pulmonary tuberculosis have reported poor sensitivity and/or specificity. We have developed a procedure in which mycobacterial DNA in crude samples is specifically captured prior to amplification, thereby concentrating the target sequences and removing irrelevant DNA and other inhibitors of the amplification reaction (sequence capture PCR). To evaluate the usefulness of this approach in the diagnosis of paucibacillary forms of pulmonary tuberculosis, sequence capture PCR was performed prospectively on samples of bronchoalveolar lavage fluid from consecutive patients suspected of having pulmonary tuberculosis but for whom three consecutive samples of respiratory secretions were smear negative. Of the 27 patients evaluated, active tuberculosis was diagnosed in nine; sequence capture PCR was positive for all of these patients, including the three for whom all specimens submitted for culture were negative. No positive results were obtained for lavage fluid from the 18 patients for whom the diagnosis of active tuberculosis was subsequently excluded or 25 additional patients undergoing bronchoalveolar lavage for evaluation of other pulmonary problems, even though many of these patients had a history of prior tuberculosis or radiographic evidence of prior tuberculous infection. Paucibacillary forms of pulmonary tuberculosis can be rapidly identified with high sensitivity and specificity using sequence capture PCR performed on samples obtained by bronchoalveolar lavage.
基于聚合酶链反应（PCR）的技术可用于快速从结核病患者的临床样本中鉴定结核分枝杆菌的DNA，但先前评估该方法在诊断少菌型肺结核方面的研究报告称其敏感性和/或特异性较差。我们开发了一种程序，在扩增之前特异性捕获粗样本中的分枝杆菌DNA，从而浓缩靶序列并去除无关DNA和扩增反应的其他抑制剂（序列捕获PCR）。为了评估该方法在诊断少菌型肺结核中的实用性，我们对连续怀疑患有肺结核但连续三份呼吸道分泌物涂片均为阴性的患者的支气管肺泡灌洗液样本进行了前瞻性序列捕获PCR检测。在评估的27例患者中，9例被诊断为活动性肺结核；序列捕获PCR对所有这些患者均呈阳性，包括所有送检培养的标本均为阴性的3例患者。对于随后排除活动性肺结核诊断的18例患者的灌洗液，以及另外25例因评估其他肺部问题而接受支气管肺泡灌洗的患者，均未获得阳性结果，尽管这些患者中有许多人有既往结核病史或既往结核感染的影像学证据。使用对通过支气管肺泡灌洗获得的样本进行的序列捕获PCR，可以高敏感性和特异性快速鉴定少菌型肺结核。
