Higgins Owen, Clancy Eoin, Forrest Matthew S, Piepenburg Olaf, Cormican Martin, Boo Teck Wee, O'Sullivan Nicola, McGuinness Claire, Cafferty Deirdre, Cunney Robert, Smith Terry J
Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland.
Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland.
Anal Biochem. 2018 Apr 1;546:10-16. doi: 10.1016/j.ab.2018.01.016. Epub 2018 Jan 31.
Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technology that provides rapid and robust infectious disease pathogen detection, ideal for point-of-care (POC) diagnostics in disease-prevalent low-resource countries. We have developed and evaluated three duplex RPA assays incorporating competitive internal controls for the detection of leading bacterial meningitis pathogens. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae singleplex RPA assays were initially developed and evaluated, demonstrating 100% specificity with limits of detection of 4.1, 8.5 and 3.9 genome copies per reaction, respectively. Each assay was further developed into internally controlled duplex RPA assays via the incorporation of internal amplification control templates. Clinical performance of each internally controlled duplex RPA assay was evaluated by testing 64 archived PCR-positive clinical samples. Compared to real-time PCR, all duplex RPA assays demonstrated 100% diagnostic specificity, with diagnostic sensitivities of 100%, 86.3% and 100% for the S. pneumoniae, N. meningitidis and H. influenzae assays, respectively. This study details the first report of internally controlled duplex RPA assays for the detection of bacterial meningitis pathogens: S. pneumoniae, N. meningitidis and H. influenzae. We have successfully demonstrated the clinical diagnostic utility of each duplex RPA assay, introducing effective diagnostic technology for POC bacterial meningitis identification in disease-prevalent developing countries.
重组酶聚合酶扩增（RPA）是一种等温核酸扩增技术，可快速、可靠地检测传染病病原体，非常适合在疾病流行的资源匮乏国家进行即时检测（POC）诊断。我们开发并评估了三种包含竞争性内部对照的双链RPA检测方法，用于检测主要的细菌性脑膜炎病原体。最初开发并评估了肺炎链球菌、脑膜炎奈瑟菌和流感嗜血杆菌的单链RPA检测方法，其特异性均为100%，检测限分别为每个反应4.1、8.5和3.9个基因组拷贝。通过加入内部扩增对照模板，将每种检测方法进一步开发为内部对照双链RPA检测方法。通过检测64份存档的PCR阳性临床样本，评估了每种内部对照双链RPA检测方法的临床性能。与实时PCR相比，所有双链RPA检测方法的诊断特异性均为100%，肺炎链球菌、脑膜炎奈瑟菌和流感嗜血杆菌检测方法的诊断敏感性分别为100%、86.3%和100%。本研究详细报道了用于检测细菌性脑膜炎病原体（肺炎链球菌、脑膜炎奈瑟菌和流感嗜血杆菌）的内部对照双链RPA检测方法。我们成功地证明了每种双链RPA检测方法的临床诊断效用，为疾病流行的发展中国家即时诊断细菌性脑膜炎引入了有效的诊断技术。
