Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by loop-mediated isothermal amplification (LAMP).
Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by loop-mediated isothermal amplification (LAMP).
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DOI：
10.1371/journal.pntd.0001249
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发表时间：
2011-08
影响因子：
3.8
通讯作者：
Dumler JS
中科院分区：
文献类型：
--
作者：
Grab DJ;Nikolskaia OV;Inoue N;Thekisoe OM;Morrison LJ;Gibson W;Dumler JS
环介导等温扩增（LAMP）法具有简单、快速和成本效益的优点，已发展成为检测包括非洲锥虫在内的多种病原微生物的最敏感和最特异的方法之一。虽然许多基于lamp的检测方法对检测远低于单个寄生虫中存在的量的DNA足够敏感，但该检测方法的检测限受检测样品体积中存在的寄生虫数量的限制；即1 /µL或103 / mL。我们假设，通过在LAMP检测前简单地向样品中添加洗涤剂，可以接近甚至获得模仿寄生虫DNA分析极限的临床敏感性。为了证明原理，我们使用了两种不同的LAMP测定法，能够检测0.1 fg基因组DNA（0.001寄生虫）。在人脑脊液（CSF）或血液中添加或不添加Triton X-100洗涤剂并在室温下孵育60分钟，对布氏罗得西亚锥虫的完整血流稀释系列进行了检测。以人脑脊液为模板，在不添加洗涤剂的情况下，以1µL脑脊液为模板，LAMP对活的完整寄生虫的检出限最高为103只/mL。值得注意的是，洗涤剂增强LAMP测定的灵敏度降低了约100至1000倍；即10比1寄生虫/mL。使用从滤纸卡片中提取的DNA，在卡片点状前用洗涤剂预处理过的血液或在洗涤剂预处理过的卡片上点状的血液样本，也发现了类似的洗涤剂介导的LAMP分析灵敏度的增加。这一通过LAMP加强对生物体液中活的非洲锥虫的检测的简单程序，为使LAMP适应于经济和敏感地诊断导致困扰发展中世界的疾病的其他原生动物寄生虫和微生物铺平了道路。非洲人类锥虫病或昏睡病是一种致命疾病（如果未经治疗），通过吸血采采蝇传播。锥虫首先进入血液和淋巴，最终侵入大脑。在农村临床环境中，诊断仍然依赖于显微镜检测血液和脑脊液（CSF）中的这些微生物。LAMP，或环介导的DNA等温扩增，是一种可以从生物体中特异性检测极少量DNA的技术。它类似于PCR，聚合酶链反应，另一种广泛用于传染病诊断的DNA扩增技术。LAMP的优点是反应在一个温度下进行，而PCR需要一个热循环器，而且LAMP不受抑制PCR的血液成分的影响。我们的研究表明，在样品制备过程中简单地加入洗涤剂，LAMP针对许多基因拷贝的分析灵敏度大大提高，这可能是因为DNA从病原体细胞中释放出来并分散在样品中。为了证明原理，我们在不同的人体体液（脑脊液或血液）中使用了致病性锥虫，但这种简单的修改应该适用于其他微生物感染的诊断，其中细胞对洗涤剂溶解敏感。
The 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. For 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. This 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. Human African trypanosomiasis or sleeping sickness is a fatal disease (if untreated) spread by bloodsucking tsetse flies. Trypanosome parasites first enter the blood and lymph and eventually invade the brain. In rural clinical settings, diagnosis still relies on the detection of these microbes in blood and cerebrospinal fluid (CSF) by microscopy. LAMP, or loop-mediated isothermal amplification of DNA, is a technique that can specifically detect very small amounts of DNA from an organism. It is similar to PCR, the polymerase chain reaction, another DNA amplification technique widely used for diagnosis of infectious diseases. LAMP's advantages are that the reaction works at one temperature, whereas PCR needs a thermocycler, and LAMP is not affected by blood components that inhibit PCR. We show that by simply adding detergent during sample preparation, the analytical sensitivity of LAMP targeting many gene copies is greatly improved, presumably because DNA is released from the pathogen cells and dispersed through the sample. To demonstrate proof of principle, we used pathogenic trypanosomes in different human body fluids (CSF or blood), but this simple modification should be applicable for diagnosis of other microbial infections where cells are sensitive to detergent lysis.
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