Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay.
Mobile suitcase laboratory for rapid detection of Leishmania donovani using recombinase polymerase amplification assay.
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DOI：
10.1186/s13071-016-1572-8
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发表时间：
2016-05-13
影响因子：
3.2
通讯作者：
Abd El Wahed A
中科院分区：
文献类型：
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作者：
Mondal D;Ghosh P;Khan MA;Hossain F;Böhlken-Fascher S;Matlashewski G;Kroeger A;Olliaro P;Abd El Wahed A
杜氏利什曼原虫（LD）是一种由白蛉传播给人类的原虫，可引起内脏利什曼病（VL）。目前，诊断是基于抗LD抗体和临床症状的存在。分子诊断需要实时PCR，这在现场环境中不容易实施。在这项研究中，我们报告的发展和测试的重组酶聚合酶扩增（RPA）检测LD。应用基因组DNA样品测定测定分析灵敏度。用利什曼原虫DNA检测试剂盒的交叉反应性。和考虑用于鉴别诊断的病原体。在LD阳性和阴性样本上评价了检测试剂盒的临床性能。所有结果均与实时PCR进行比较。为了允许在现场环境中使用该检测试剂盒，在孟加拉国Mymensingh的当地医院开发并运行了一个移动的手提箱实验室（56 × 45.5 × 26.5 cm）。LD RPA检测试剂盒检测到相当于一个LD基因组DNA。在恒温（42 °C）下在15分钟内进行测定。RPA检测还检测到其他利什曼原虫属（L.梅杰湖aethiopica和L. infantum），但未鉴定出其他病原体的核酸。来自VL、无症状和黑热病后皮肤利什曼病受试者的48个样品通过LD RPA和实时PCR测定检测为阳性，48个LD阴性样品为阴性，这表明100%一致。手提箱实验室在当地医院成功地使用了太阳能电池。通过一种新的基于磁珠的方法（SpeedXtract）进行DNA提取，其中应用了简单的快速裂解方案。所有试剂均不依赖冷链。使用RPA的移动的手提箱实验室是快速、敏感和特异性检测LD的理想选择，尤其是在低资源环境下，并有助于VL控制和消除方案。本文的在线版本（doi：10.1186/s13071-016-1572-8）包含补充材料，可供授权用户使用。
Leishmania donovani (LD) is a protozoan parasite transmitted to humans from sand flies, which causes Visceral Leishmaniasis (VL). Currently, the diagnosis is based on presence of the anti-LD antibodies and clinical symptoms. Molecular diagnosis would require real-time PCR, which is not easy to implement at field settings. In this study, we report on the development and testing of a recombinase polymerase amplification (RPA) assay for the detection of LD. A genomic DNA sample was applied to determine the assay analytical sensitivity. The cross-reactivity of the assay was tested by DNA of Leishmania spp. and of pathogens considered for differential diagnosis. The clinical performance of the assay was evaluated on LD positive and negative samples. All results were compared with real-time PCR. To allow the use of the assay at field settings, a mobile suitcase laboratory (56 × 45.5 × 26.5 cm) was developed and operated at the local hospital in Mymensingh, Bangladesh. The LD RPA assay detected equivalent to one LD genomic DNA. The assay was performed at constant temperature (42 °C) in 15 min. The RPA assay also detected other Leishmania species (L. major, L. aethiopica and L. infantum), but did not identify nucleic acid of other pathogens. Forty-eight samples from VL, asymptomatic and post-kala-azar dermal leishmaniasis subjects were detected positive and 48 LD-negative samples were negative by both LD RPA and real-time PCR assays, which indicates 100 % agreement. The suitcase laboratory was successfully operated at the local hospital by using a solar-powered battery. DNA extraction was performed by a novel magnetic bead based method (SpeedXtract), in which a simple fast lysis protocol was applied. Moreover, All reagents were cold-chain independent. The mobile suitcase laboratory using RPA is ideal for rapid sensitive and specific detection of LD especially at low resource settings and could contribute to VL control and elimination programmes. The online version of this article (doi:10.1186/s13071-016-1572-8) contains supplementary material, which is available to authorized users.