Ruiz-Fuentes Jenny Laura, Díaz Alexis, Entenza Anayma Elena, Frión Yahima, Suárez Odelaisy, Torres Pedro, de Armas Yaxsier, Acosta Lucrecia
National Reference Laboratory for Tuberculosis, Leprosy and other Mycobacterial Diseases, Department of Microbiology, "Pedro Kourí" Tropical Medicine Institute, Havana, Cuba.
Laboratory of Chemicals and Biopharmaceuticals (LABIOFAM), Havana, Cuba.
Int J Mycobacteriol. 2015 Dec;4(4):284-9. doi: 10.1016/j.ijmyco.2015.06.005. Epub 2015 Jul 15.
OBJECTIVE/BACKGROUND: The diagnosis of leprosy has been a challenge due to the low sensibility of the conventional methods and the impossibility of culturing the causative organism. In this study, four methods for Mycobacterium leprae nucleic-acid extraction from Ziehl-Neelsen-stained slides (ZNS slides) were compared: Phenol/chloroform, Chelex 100 resin, and two commercial kits (Wizard Genomic DNA Purification Kit and QIAamp DNA Mini Kit).
DNA was extracted from four groups of slides: a high-codification-slide group (bacteriological index [BI]⩾4), a low-codification-slide group (BI=1), a negative-slide group (BI=0), and a negative-control-slide group (BI=0). Quality DNA was evidenced by the amplification of specific repetitive element present in M. leprae genomic DNA (RLEP) using a nested polymerase chain reaction.
This is the first report comparing four different extraction methods for obtaining M. leprae DNA from ZNS slides in Cuban patients, and applied in molecular diagnosis. Good-quality DNA and positive amplification were detected in the high-codification-slide group with the four methods, while from the low-codification-slide group only the QIAGEN and phenol-chloroform methods obtained amplification of M. leprae. In the negative-slide group, only the QIAGEN method was able to obtain DNA with sufficient quality for positive amplification of the RLEP region. No amplification was observed in the negative-control-slide group by any method. Patients with ZNS negative slides can still transmit the infection, and molecular methods can help identify and treat them, interrupting the chain of transmission and preventing the onset of disabilities.
The ZNS slides can be sent easily to reference laboratories for later molecular analysis that can be useful not only to improve the diagnosis, but also for the application of other molecular techniques.
目的/背景：由于传统方法的敏感性较低且无法培养致病生物体，麻风病的诊断一直是一项挑战。在本研究中，比较了从萋-尼染色玻片（ZNS玻片）中提取麻风分枝杆菌核酸的四种方法：苯酚/氯仿法、Chelex 100树脂法以及两种商业试剂盒（Wizard基因组DNA纯化试剂盒和QIAamp DNA微量试剂盒）。
从四组玻片提取DNA：高编码玻片组（细菌学指数[BI]⩾4）、低编码玻片组（BI = 1）、阴性玻片组（BI = 0）和阴性对照玻片组（BI = 0）。使用巢式聚合酶链反应扩增麻风分枝杆菌基因组DNA中存在的特定重复元件（RLEP）来证明高质量DNA的存在。
这是第一份比较从古巴患者的ZNS玻片中获取麻风分枝杆菌DNA的四种不同提取方法并应用于分子诊断的报告。在高编码玻片组中，用这四种方法均检测到了高质量DNA和阳性扩增，而在低编码玻片组中，只有QIAGEN法和苯酚-氯仿法获得了麻风分枝杆菌的扩增。在阴性玻片组中，只有QIAGEN法能够获得质量足以对RLEP区域进行阳性扩增的DNA。任何方法在阴性对照玻片组中均未观察到扩增。ZNS玻片为阴性的患者仍可传播感染，分子方法有助于识别和治疗他们，从而中断传播链并预防残疾的发生。
ZNS玻片可以很容易地送到参考实验室进行后续分子分析，这不仅有助于改善诊断，还可用于其他分子技术的应用。
