Rapid detection of Mycobacterium ulcerans with isothermal recombinase polymerase amplification assay
Rapid detection of Mycobacterium ulcerans with isothermal recombinase polymerase amplification assay
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DOI：
10.1371/journal.pntd.0007155
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发表时间：
2019-02-01
影响因子：
3.8
通讯作者：
Phillips, Richard Odame
中科院分区：
文献类型：
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作者：
Frimpong, Michael;Ahor, Hubert Senanu;Phillips, Richard Odame
背景根据世界卫生组织的说法，获得布鲁里溃疡的准确诊断测试是一个研究重点。聚合酶链反应（PCR）核酸扩增插入序列IS2404是检测溃疡分枝杆菌（Mycobacterium ulcerans， M. ulcerans）最敏感、最特异的方法。然而，由于成本和技术的复杂性，聚合酶链反应在非洲的流行社区并不总是可用。等温DNA扩增系统，如重组酶聚合酶扩增（RPA）已经成为一种分子诊断工具，具有与PCR相似的准确性，但具有在恒定的较低温度下在较短时间内扩增模板DNA的优势。本研究的目的是开发用于溃疡分枝杆菌检测的RPA，并评估其在布鲁里溃疡疾病中的应用。采用RPA法，在42℃恒温条件下，在15分钟内扩增出溃疡分枝杆菌IS2404的特定片段。每次反应的检出限为45份IS2404分子DNA标准品。7株溃疡分枝杆菌均可检出，特异性高，与其他分枝杆菌及临床相关菌种均无交叉反应。溃疡分枝杆菌（Mu-RPA）检测的临床表现是通过从67例怀疑布鲁里溃疡患者和12例临床证实非布鲁里溃疡病变患者的细针抽吸或拭子中提取DNA来评估的。所有结果与高灵敏度实时PCR进行比较。Mu-RPA检测的临床特异性为100% (95% CI, 84-100)，敏感性为88% （95% CI, 77-95）。结论Mu-RPA分析是PCR的一种替代方法，特别是在基础设施有限的地区。在大多数溃疡分枝杆菌流行的国家，目前检测溃疡分枝杆菌的诊断方法由于将样品运送和储存到遥远的集中实验室的时间较长，因而产生结果的时间较长。溃疡分枝杆菌重组酶聚合酶扩增试验（Mu-RPA）是一种新的快速诊断试验，用于检测溃疡分枝杆菌感染，通常称为布鲁里溃疡，是一种慢性、衰弱性、坏死性皮肤和软组织疾病。这种检测方法适合在便携式检测设备上使用，有可能在需要时用于快速诊断，向布鲁里溃疡治疗诊所的卫生工作者提供及时的结果。
Background Access to an accurate diagnostic test for Buruli ulcer (BU) is a research priority according to the World Health Organization. Nucleic acid amplification of insertion sequence IS2404 by polymerase chain reaction (PCR) is the most sensitive and specific method to detect Mycobacterium ulcerans (M. ulcerans), the causative agent of BU. However, PCR is not always available in endemic communities in Africa due to its cost and technological sophistication. Isothermal DNA amplification systems such as the recombinase polymerase amplification (RPA) have emerged as a molecular diagnostic tool with similar accuracy to PCR but having the advantage of amplifying a template DNA at a constant lower temperature in a shorter time. The aim of this study was to develop RPA for the detection of M. ulcerans and evaluate its use in Buruli ulcer disease.Methodology and principal findings A specific fragment of IS2404 of M. ulcerans was amplified within 15 minutes at a constant 42 degrees C using RPA method. The detection limit was 45 copies of IS2404 molecular DNA standard per reaction. The assay was highly specific as all 7 strains of M. ulcerans tested were detected, and no cross reactivity was observed to other mycobacteria or clinically relevant bacteria species. The clinical performance of the M. ulcerans (Mu-RPA) assay was evaluated using DNA extracted from fine needle aspirates or swabs taken from 67 patients in whom BU was suspected and 12 patients with clinically confirmed non-BU lesions. All results were compared to a highly sensitive real-time PCR. The clinical specificity of the Mu-RPA assay was 100% (95% CI, 84-100), whiles the sensitivity was 88% (95% CI, 77-95).Conclusion The Mu-RPA assay represents an alternative to PCR, especially in areas with limited infrastructure.Author summary Current diagnostic methods to detect M. ulcerans suffer from delayed time-to-results in most endemic countries by the prolonged period of time for the shipment and storage of samples to a distant, centralized laboratory. The M. ulcerans recombinase polymerase amplification assay (Mu-RPA) is a new, rapid diagnostic test developed for the detection of M. ulcerans infection, known commonly as Buruli ulcer, a chronic, debilitating, necrotizing disease of the skin and soft tissues. This assay is suitable for use on a portable detection device, with the potential to be used for quick diagnosis at the point of need, providing timely results to health workers at Buruli ulcer treatment clinics.