Centre for Infectious Diseases, Division of Pathway Medicine, School of Biomedical Sciences, College of Medicine and Veterinary Medicine, University of Edinburgh, Edinburgh, United Kingdom.
PLoS Negl Trop Dis. 2010 Nov 2;4(11):e865. doi: 10.1371/journal.pntd.0000865.
Loop-mediated isothermal amplification (LAMP) is at the forefront of the search for innovative diagnostics for human African trypanosomiasis (HAT). Several simple endpoint detection methods have been developed for LAMP and here we compare four of these: (i) visualization of turbidity; (ii) addition of hydroxynaphthol blue before incubation; (iii) addition of calcein with MnCl₂ before incubation and (iv) addition of Quant-iT PicoGreen after incubation. These four methods were applied to four LAMP assays for the detection of human African trypanosomiasis, including two Trypanozoon specific and two Trypanosoma brucei rhodesiense specific reactions using DNA extracted from cryo-preserved procyclic form T. b. rhodesiense. A multi-observer study was performed to assess inter-observer reliability of two of these methods: hydroxynapthol blue and calcein with MnCl₂, using DNA prepared from blood samples stored on Whatman FTA cards. Results showed that hydroxynaphthol blue was the best of the compared methods for easy, inexpensive, accurate and reliable interpretation of LAMP assays for HAT. Hydroxynapthol blue generates a violet to sky blue colour change that was easy to see and was consistently interpreted by independent observers. Visible turbidity detection is not possible for all currently available HAT LAMP reactions; Quant-iT PicoGreen is expensive and addition of calcein with MnCl₂ adversely affects reaction sensitivity and was unpopular with several observers.
环介导等温扩增（LAMP）是寻找创新性人类非洲锥虫病（HAT）诊断方法的前沿领域。已经开发出几种用于 LAMP 的简单终点检测方法，在这里我们比较了其中的四种：（i）浊度的可视化；（ii）在孵育前添加羟基萘酚蓝；（iii）在孵育前添加钙黄绿素和 MnCl₂；（iv）孵育后添加 Quant-iT PicoGreen。这四种方法应用于四种用于检测人类非洲锥虫病的 LAMP 检测方法，包括两种针对 Trypanozoon 特异性的反应和两种针对 Trypanosoma brucei rhodesiense 特异性的反应，使用从冷冻保存的前鞭毛体 T. b. rhodesiense 中提取的 DNA。进行了一项多观察者研究，以评估两种方法（羟基萘酚蓝和钙黄绿素与 MnCl₂）的观察者间可靠性，这些方法使用从储存在 Whatman FTA 卡上的血液样本中提取的 DNA。结果表明，羟基萘酚蓝是比较方法中用于 HAT 的 LAMP 检测的简便、经济、准确和可靠解释的最佳方法。羟基萘酚蓝产生从紫色到天蓝色的颜色变化，易于观察，并且被独立观察者一致解释。目前可用于所有 HAT LAMP 反应的可见浊度检测不可行；Quant-iT PicoGreen 昂贵，添加钙黄绿素和 MnCl₂会降低反应灵敏度，并且不受几位观察者的欢迎。
