Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland.
School of Medicine, National University of Ireland, Galway and Galway University Hospital, Galway, Ireland.
Int J Mol Sci. 2018 Feb 9;19(2):524. doi: 10.3390/ijms19020524.
Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: , and . Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for , and of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of endonuclease IV incorporation into nucleic acid amplification diagnostic technology.
细菌性脑膜炎感染是一个主要的全球健康关注点，快速准确的诊断对于降低相关发病率和死亡率至关重要。环介导等温扩增（LAMP）提供了一种有效的低成本诊断方法；然而，多重 LAMP 难以实现，限制了其应用。我们使用内切核酸酶 IV 和独特的 LAMP 引物/探针开发了新型实时多重 LAMP 技术 TEC-LAMP。本研究评估了一种内部对照的多重 TEC-LAMP 检测方法用于检测主要细菌性脑膜炎病原体的分析特异性、检测限（LOD）和临床应用： 、 和 。通过测试 168 株细菌菌株来建立分析特异性，使用 Probit 分析来确定 LOD。TEC-LAMP 检测方法具有 100%的特异性，对于 、 和 的 LOD 分别为 39.5、17.3 和 25.9 个基因组拷贝/反应。通过测试 65 个存档的 PCR 阳性样本来评估临床性能。与单重实时 PCR 相比，多重 TEC-LAMP 检测方法的诊断灵敏度和特异性分别为 92.3%和 100%。这是首次报道用于细菌性脑膜炎病原体检测的单管内部对照多重 LAMP 检测方法，也是首次报道将内切核酸酶 IV 纳入核酸扩增诊断技术。
