建立一种用于检测活性伊氏锥虫感染的重组酶聚合酶扩增侧向流动检测法。
Development of a recombinase polymerase amplification lateral flow assay for the detection of active Trypanosoma evansi infections.
机构信息
Research Unit for Cellular and Molecular Immunology (CMIM), Vrije Universiteit Brussel (VUB), Brussels, Belgium.
Laboratory of Medical Biochemistry and the Infla-Med Centre of Excellence, University of Antwerp (UA), Campus Drie Eiken, Wilrijk, Belgium.
出版信息
PLoS Negl Trop Dis. 2020 Feb 18;14(2):e0008044. doi: 10.1371/journal.pntd.0008044. eCollection 2020 Feb.
BACKGROUND
Animal trypanosomosis caused by Trypanosoma evansi is known as "surra" and is a widespread neglected tropical disease affecting wild and domestic animals mainly in South America, the Middle East, North Africa and Asia. An essential necessity for T. evansi infection control is the availability of reliable and sensitive diagnostic tools. While DNA-based PCR detection techniques meet these criteria, most of them require well-trained and experienced users as well as a laboratory environment allowing correct protocol execution. As an alternative, we developed a recombinase polymerase amplification (RPA) test for Type A T. evansi. The technology uses an isothermal nucleic acid amplification approach that is simple, fast, cost-effective and is suitable for use in minimally equipped laboratories and even field settings.
METHODOLOGY/PRINCIPLE FINDINGS: An RPA assay targeting the T. evansi RoTat1.2 VSG gene was designed for the DNA-based detection of T. evansi. Comparing post-amplification visualization by agarose gel electrophoresis and a lateral flow (LF) format reveals that the latter displays a higher sensitivity. The RPA-LF assay is specific for RoTat1.2-expressing strains of T. evansi as it does not detect the genomic DNA of other trypanosomatids. Finally, experimental mouse infection trials demonstrate that the T. evansi specific RPA-LF can be employed as a test-of-cure tool.
CONCLUSIONS/SIGNIFICANCE: Compared to other DNA-based parasite detection methods (such as PCR and LAMP), the T. evansi RPA-LF (TevRPA-LF) described in this paper is an interesting alternative because of its simple read-out (user-friendly), short execution time (15 minutes), experimental sensitivity of 100 fg purified genomic T. evansi DNA, and ability to be carried out at a moderate, constant temperature (39°C). Therefore, the TevRPA-LF is an interesting tool for the detection of active T. evansi infections.
背景
由伊氏锥虫引起的动物锥虫病被称为“苏拉病”，是一种广泛存在的被忽视的热带病，主要影响南美的野生和家养动物、中东、北非和亚洲。伊氏锥虫感染控制的一个必要条件是提供可靠和敏感的诊断工具。虽然基于 DNA 的 PCR 检测技术符合这些标准，但其中大多数需要经过良好培训和经验丰富的用户，以及允许正确执行方案的实验室环境。作为替代方案，我们开发了一种用于 A 型伊氏锥虫的重组酶聚合酶扩增（RPA）测试。该技术使用一种简单、快速、具有成本效益的等温核酸扩增方法，适用于设备简陋的实验室，甚至是野外环境。
方法/原理发现：针对伊氏锥虫 RoTat1.2 VSG 基因设计了一种 RPA 检测方法，用于 DNA 检测伊氏锥虫。通过琼脂糖凝胶电泳和侧向流动（LF）格式的扩增后可视化比较，后者显示出更高的灵敏度。RPA-LF 检测方法特异性针对表达 RoTat1.2 的伊氏锥虫菌株，因为它不能检测其他锥虫的基因组 DNA。最后，实验性小鼠感染试验表明，T. evansi 特异性 RPA-LF 可作为治愈试验工具。
结论/意义：与其他基于 DNA 的寄生虫检测方法（如 PCR 和 LAMP）相比，本文所述的伊氏锥虫 RPA-LF（TevRPA-LF）是一种有趣的替代方法，因为它的读取简单（用户友好）、执行时间短（15 分钟）、实验灵敏度为 100 fg 纯化的基因组 T. evansi DNA，以及在中等、恒定温度（39°C）下进行的能力。因此，TevRPA-LF 是检测活动性伊氏锥虫感染的一种有趣工具。
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