Lucchi Naomi W, Ljolje Dragan, Silva-Flannery Luciana, Udhayakumar Venkatachalam
Malaria Branch, Division of Parasitic Diseases and Malaria, Center for Global Health, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America.
Atlanta Research and Education Foundation/Veterans Affairs Medical center, Decatur Georgia, United States of America.
PLoS One. 2016 Mar 11;11(3):e0151437. doi: 10.1371/journal.pone.0151437. eCollection 2016.
Malaria elimination efforts are hampered by the lack of sensitive tools to detect infections with low-level parasitemia, usually below the threshold of standard diagnostic methods, microscopy and rapid diagnostic tests. Isothermal nucleic acid amplification assays such as the loop-mediated isothermal amplification (LAMP), are well suited for field use as they do not require thermal cyclers to run the test. However, the use of specialized equipment, as described by many groups, reduces the versatility of the LAMP technique as a simple tool for use in endemic countries. In this study, the use of the malachite green (MG) dye, as a visual endpoint readout, together with a simple mini heat block was evaluated for the detection of malaria parasites. The assay was performed for 1 hour at 63°C and the results scored by 3 independent human readers. The limit of detection of the assay was determined using well-quantified Plasmodium spp. infected reference samples and its utility in testing clinical samples was determined using 190 pre-treatment specimens submitted for reference diagnosis of imported malaria in the United States. Use of a simplified boil and spin methods of DNA extraction from whole blood and filter paper was also investigated. We demonstrate the accurate and sensitive detection of malaria parasites using this assay with a detection limit ranging between 1-8 parasites/μL, supporting its applicability for the detection of infections with low parasite burden. This assay is compatible with the use of a simple boil and spin sample preparation method from both whole blood and filter papers without a loss of sensitivity. The MG-LAMP assay described here has great potential to extend the reach of molecular tools to settings where they are needed.
疟疾消除工作受到缺乏敏感工具的阻碍，这些工具用于检测低水平寄生虫血症感染，通常低于标准诊断方法（显微镜检查和快速诊断测试）的阈值。等温核酸扩增检测法，如环介导等温扩增（LAMP），非常适合现场使用，因为它们不需要热循环仪来进行检测。然而，正如许多研究小组所描述的那样，使用专门设备降低了LAMP技术作为流行国家简单工具的通用性。在本研究中，评估了使用孔雀石绿（MG）染料作为可视化终点读数，结合简单的小型加热块来检测疟原虫。该检测在63°C下进行1小时，结果由3名独立的人员进行评分。使用定量良好的感染疟原虫属的参考样本确定该检测的检测限，并使用提交给美国进口疟疾参考诊断的190份治疗前标本确定其在检测临床样本中的效用。还研究了使用简化的煮沸和离心方法从全血和滤纸上提取DNA。我们证明了使用该检测方法能够准确、灵敏地检测疟原虫，检测限在1 - 8个寄生虫/微升之间，支持其适用于检测低寄生虫载量的感染。该检测与使用简单的煮沸和离心样本制备方法兼容，可用于全血和滤纸，且不会损失灵敏度。本文所述的MG-LAMP检测法有很大潜力将分子工具的应用范围扩展到需要的环境中。
