Santos A R, De Miranda A B, Sarno E N, Suffys P N, Degrave W M
Department of Tropical Medicine-Leprosy Sector, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil.
J Med Microbiol. 1993 Oct;39(4):298-304. doi: 10.1099/00222615-39-4-298.
DNA of Mycobacterium leprae, obtained by a highly efficient nucleic acid extraction procedure, was used for standardisation of the amplification of an M. leprae-specific repetitive sequence by use of the polymerase chain reaction (PCR). With pure DNA, M. leprae-specific amplification was obtained with as low as 100 ag (1 ag = 10(-18) g) of target DNA, a quantity equal to about one-tenth of the bacterial genome. Optimal processing of different types of clinical samples such as biopsy material, blood and lymph fluid, from multibacillary leprosy patients, was studied. Simple freezing-boiling cycles in the presence of Triton X100, with some additional sample-specific modifications such as pre-treatment with NaOH to eliminate PCR inhibitors, was found to be sufficient to yield amplification of bacterial DNA in samples from paucibacillary patients. Clinical samples from 27 untreated leprosy patients, covering the various clinical forms of the disease, and with a bacterial index ranging from 5+ to 0, were collected and processed for PCR analysis. After hybridisation of the amplified material with a specific sequence, 25 of 27 patients analysed gave positive results for M. leprae in at least one of the samples. The potential of PCR for the diagnosis of leprosy is discussed.
通过高效核酸提取程序获得的麻风分枝杆菌DNA，用于通过聚合酶链反应（PCR）对麻风分枝杆菌特异性重复序列的扩增进行标准化。使用纯DNA时，低至100 ag（1 ag = 10^(-18) g）的靶DNA即可获得麻风分枝杆菌特异性扩增，该量约等于细菌基因组的十分之一。研究了对多菌型麻风患者不同类型临床样本（如活检材料、血液和淋巴液）的最佳处理方法。发现在Triton X100存在下进行简单的冻融循环，并进行一些额外的样本特异性修饰（如用NaOH预处理以消除PCR抑制剂），足以在少菌型患者的样本中扩增细菌DNA。收集了27例未经治疗的麻风患者的临床样本，涵盖了该疾病的各种临床类型，细菌指数范围从5+到0，并对其进行PCR分析处理。将扩增产物与特定序列杂交后，27例接受分析的患者中有25例在至少一个样本中给出了麻风分枝杆菌的阳性结果。讨论了PCR在麻风诊断中的潜力。
