优化结核分枝杆菌检测的等温扩增方法和野外工作的可视化方法。
Optimization of isothermal amplification method for Mycobacteriumtuberculosisdetection and visualization method for fieldwork.
机构信息
Materials Institute, TÜBİTAK Marmara Research Center, Kocaeli, Turkey
Department of Medical Laboratory Techniques, Vocational School of Health Services, İstanbul Medipol University, İstanbul, Turkey
出版信息
Turk J Med Sci. 2020 Jun 23;50(4):1069-1075. doi: 10.3906/sag-1910-6.
BACKGROUND/AIM: Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure.
MATERIALS AND METHODS
Sputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA.DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB’s Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used.
RESULTS
Optimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method was 102 CFU/mL and sensitivity was determined 100% compared to five different Mycobacterium species.
CONCLUSION
The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.
背景/目的：结核病仍然是全球最具传染性的疾病之一。结核病控制的关键因素是快速诊断、有效治疗以及通过监测和监控防止污染。然而，培养是结核病实验室诊断的金标准方法；结果需要数周时间才能获得。为了防止结核病的污染，诊断必须在短时间内进行，并且应尽快开始治疗。本研究的目的是优化环介导等温扩增（LAMP）方法，该方法比聚合酶链反应（PCR）方法提供更快、更敏感的结果，并允许在等温条件下复制靶核酸序列，而无需实验室基础设施。
材料和方法
将痰液样本与 5%氯化钙中的胰蛋白酶溶液混合，以获得 DNA。使用 QIAGEN QIAamp DNA 迷你试剂盒纯化 DNA。根据结核分枝杆菌的 IS6110 基因，使用 Primer explorer V5 程序设计 LAMP 引物。使用 NEB Bst 3.0 DNA 聚合酶试剂盒进行 LAMP 反应。此外，使用 NEB 的 Taq 聚合酶试剂盒，将 LAMP 反应与基于 TaqMan 的 RT-PCR 方法进行比较。最后，为了可视化 LAMP 产物，使用 Milenia Biotec 生产的侧向流动试纸、NEB 生产的比色 2X LAMP 主混合物和 2%w/v 琼脂糖凝胶电泳方法。
结果
发现 LAMP 的最佳扩增温度为 71.4°C。该方法的检测限为 102 CFU/mL，与五种不同的分枝杆菌属相比，灵敏度为 100%。
结论
本研究表明，优化后的痰液样本 LAMP-LFD 和比色 LAMP 方案可作为一种快速、敏感和特异的现场结核病诊断方法。
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