Goyal M, Lawn S, Afful B, Acheampong J W, Griffin G, Shaw R
Department of Respiratory Medicine, NHLI, Imperial College School of Medicine, London, UK.
J Infect. 1999 May;38(3):171-5. doi: 10.1016/s0163-4453(99)90246-3.
Molecular epidemiological studies of Mycobacterium tuberculosis in high prevalence areas in sub-Saharan Africa are hampered by the difficulty of culturing organisms from clinical samples. This study aimed to evaluate for application in a developing country, a modification of a novel polymerase chain reaction (PCR) based molecular epidemiological typing method, termed spoligotyping.
DNA extraction from sputum was followed by PCR amplification of spacers between direct repeats in the M. tuberculosis genome, and hybridization to a range of the 53 known spacer sequences.
Sputum from 175 patients in the Ashanti region of Ghana were collected, and satisfactory spoligotyping results were obtained in 159. A total of 100 different spoligotype patterns were observed with 84 patients having unique patterns and the remainder falling into 16 clusters. A number of epidemiologically linked cases were shown to be unrelated on the basis of different spoligotype patterns, but epidemiological links were not found to explain clusters. Comparison of spoligotyping of DNA extracted from sputum with restriction fragment length polymorphism (RFLP) from mycobacterial culture in a subset of 25 patients, indicated that spoligotyping was less discriminatory than RFLP, Sixteen spoligotype patterns were shown to comprise 2 3 different RFLP patterns.
This study suggests that the PCR based technique of spoligotyping can be applied successfully to DNA extracted from sputum collected in the setting of a developing country, but that this is less discriminatory than RFLP. Spoligotyping is particularly useful when used to support conventional epidemiology since a proportion of false epidemiological associations can be identified.
在撒哈拉以南非洲的结核高流行地区，结核分枝杆菌的分子流行病学研究因难以从临床样本中培养出细菌而受阻。本研究旨在评估一种基于聚合酶链反应（PCR）的新型分子流行病学分型方法（称为间隔寡核苷酸分型法）的改良方法在一个发展中国家的应用情况。
从痰中提取DNA，随后对结核分枝杆菌基因组中直接重复序列之间的间隔区进行PCR扩增，并与一系列53种已知的间隔区序列杂交。
收集了加纳阿散蒂地区175例患者的痰液，其中159例获得了满意的间隔寡核苷酸分型结果。共观察到100种不同的间隔寡核苷酸分型模式，84例患者具有独特的模式，其余患者分为16个簇。一些在流行病学上有联系的病例基于不同的间隔寡核苷酸分型模式显示并无关联，但未发现流行病学联系能解释这些簇。在25例患者的一个亚组中，对从痰中提取的DNA进行间隔寡核苷酸分型与从分枝杆菌培养物中进行限制性片段长度多态性分析（RFLP）的比较表明，间隔寡核苷酸分型的鉴别力低于RFLP。16种间隔寡核苷酸分型模式显示包含2至3种不同的RFLP模式。
本研究表明，基于PCR的间隔寡核苷酸分型技术可成功应用于从发展中国家环境中收集的痰中提取的DNA，但该方法的鉴别力低于RFLP。间隔寡核苷酸分型在用于支持传统流行病学研究时特别有用，因为可以识别出一部分错误的流行病学关联。
