Using Detergent to Enhance Detection Sensitivity of African Trypanosomes in Human CSF and Blood by Loop-Mediated Isothermal Amplification (LAMP)

BackgroundThe loop-mediated isothermal amplification (LAMP) assay, with its advantages of simplicity, rapidity and cost effectiveness, has evolved as one of the most sensitive and specific methods for the detection of a broad range of pathogenic microorganisms including African trypanosomes. While many LAMP-based assays are sufficiently sensitive to detect DNA well below the amount present in a single parasite, the detection limit of the assay is restricted by the number of parasites present in the volume of sample assayed; i.e. 1 per µL or 103 per mL. We hypothesized that clinical sensitivities that mimic analytical limits based on parasite DNA could be approached or even obtained by simply adding detergent to the samples prior to LAMP assay. Methodology/Principal FindingsFor proof of principle we used two different LAMP assays capable of detecting 0.1 fg genomic DNA (0.001 parasite). The assay was tested on dilution series of intact bloodstream form Trypanosoma brucei rhodesiense in human cerebrospinal fluid (CSF) or blood with or without the addition of the detergent Triton X-100 and 60 min incubation at ambient temperature. With human CSF and in the absence of detergent, the LAMP detection limit for live intact parasites using 1 µL of CSF as the source of template was at best 103 parasites/mL. Remarkably, detergent enhanced LAMP assay reaches sensitivity about 100 to 1000-fold lower; i.e. 10 to 1 parasite/mL. Similar detergent-mediated increases in LAMP assay analytical sensitivity were also found using DNA extracted from filter paper cards containing blood pretreated with detergent before card spotting or blood samples spotted on detergent pretreated cards. Conclusions/SignificanceThis simple procedure for the enhanced detection of live African trypanosomes in biological fluids by LAMP paves the way for the adaptation of LAMP for the economical and sensitive diagnosis of other protozoan parasites and microorganisms that cause diseases that plague the developing world.背景：环介导等温扩增（loop-mediated isothermal amplification, LAMP）技术凭借操作简便、快速高效且成本低廉的优势，已成为检测包括非洲锥虫在内的多种病原微生物的高灵敏、高特异性方法之一。尽管诸多基于LAMP的检测方法灵敏度足以检出远低于单寄生虫所含的DNA量，但该检测方法的限制受限于检测样本体积内的寄生虫数量，即每微升1个或每毫升1000个。我们提出假说：仅需在LAMP检测前向样本中添加去污剂，即可达到甚至超越基于寄生虫DNA的分析灵敏度所对应的临床灵敏度。 方法与主要结果：为验证这一原理，我们采用两种可检测0.1飞克基因组DNA（相当于0.001个寄生虫）的不同LAMP检测体系。我们以完整的罗德西亚布氏锥虫血液阶段虫体为对象，在人类脑脊液（cerebrospinal fluid, CSF）或血液中制备梯度稀释样本，分别添加或不添加去污剂Triton X-100，并在室温下孵育60分钟后开展检测。在未添加去污剂的人类脑脊液样本中，以1微升脑脊液作为模板来源时，LAMP对活完整寄生虫的检测限最高仅为1000个寄生虫/毫升。值得注意的是，添加去污剂的LAMP检测体系灵敏度提升约100至1000倍，即检测限可达10至1个寄生虫/毫升。此外，针对经去污剂预处理后点样于滤纸片的血液样本，或点样于经去污剂预处理滤纸片的血液样本所提取的DNA，同样观察到去污剂介导的LAMP分析灵敏度提升。 结论与意义：本研究提出的通过在生物体液中添加去污剂以增强LAMP检测活非洲锥虫的简便方法，为将LAMP技术适配为经济、灵敏的诊断方法奠定了基础，可用于检测其他困扰发展中国家的原生动物寄生虫及病原微生物。