基于RPA-LFD技术快速、可视化鉴别诊断隐球菌性脑膜炎与结核性脑膜炎的研究

Cryptococcal meningitis (CM) and tuberculous meningitis (TBM) show similar clinical symptoms in the early stage of the disease and are prone to be misdiagnosis. The sensitivity of cerebrospinal fluid smear microscopy is low and the false negative rate is high. Culture and identification of pathogen is long circle, high requirement and easy to delay diagnosis. At present, there is still a lack of advanced detection method for the differential diagnosis of CM and TBM. Recombinase polymerase amplification combined with lateral flow dipsticks (RPA-LFD) can visually detect target fragments and has the characteristics of rapid, simple, high sensitivity and strong specificity, et al. In our earlier study, we have introduced a rapid and visual detection method of Mycobacterium tuberculosis based on RPA-LFD technology. The RPA-LFD assay, read results with naked eye, was able to detect as little as 25 fg of genomic DNA and had no cross-reaction with other control bacteria. This project is planned to continue to study the RPA-LFD technique to detect Cryptococcus neoformans. And then, the results will be combined with that of previous study to form simultaneous rapid detection system of Cryptococcus neoformans and Mycobacterium tuberculosis and build visual differential diagnosis system of CM and TBM. Besides, the clinical diagnostic value of the system will be evaluated by detecting a large number of clinical samples. Our study will provide solid experimental basis for the research and development of diagnostic reagents or equipment and new technical method for the visual diagnosis of other infectious diseases. Our study will be helpful in the diagnosis of suspected cases of meningitis and in the clinical treatment of related diseases.

隐球菌性脑膜炎（CM）与结核性脑膜炎（TBM）在疾病初期表现出相似的临床症状，易于错误诊断。脑脊液涂片镜检敏感性低、假阴性率高；病原菌培养周期长、要求高，易于延误诊断；目前二者仍缺乏高效的鉴别诊断方法。重组酶聚合酶扩增联合侧向流试纸条（RPA-LFD）技术能够可视化检测目标核酸片段，具有快速、简便、灵敏度高、特异性强等特点。我们前期已经建立基于RPA-LFD技术的结核杆菌检测方法，裸眼判读结果，5 min快速扩增，检测限达25 fg DNA，对结核杆菌有100%特异性。本项目拟继续研究RPA-LFD技术检测隐球菌的实验方法，融合前期结果，形成新型隐球菌与结核杆菌的同步快速检测体系、建立CM与TBM的可视化鉴别诊断方法，并检测临床样本以评估该体系的诊断价值，为研发相关诊断试剂或设备提供坚实的实验基础，为其他感染性疾病的可视化诊断提供新的技术手段，有效帮助脑膜炎疑似病例的确诊及相关疾病的治疗。

按照研究计划，本项目已经建立基于重组酶聚合酶扩增联合侧向流试纸条（RPA-LFD）技术的隐球菌检测方法，裸眼判读结果，10 min完成反应，新型隐球菌检测限达到0.64 pg基因组DNA（约合30个基因组拷贝）、格特隐球菌检测限达到1.016 pg基因组DNA（约合50个基因组拷贝），对于隐球菌有100%特异性，在25-45°C温度范围内均能高效扩增，其检测临床样本（n=114）的敏感度和特异度分别为95.2%、95.8%，与目前临床广泛应用的隐球菌侧向流检测方法“CrAg Lateral Flow Assay”水平相当。同时，我们比较了两种真菌基因组提取方法：TIANamp Yeast DNA Kit“离心柱法”——得到相对纯净的基因组DNA和glass beads“玻璃珠法”——得到菌体裂解液，二者产出的扩增模板对于本检测体系的结果并无明显影响，表明此体系的耐受性较强。此外，我们回顾性分析了2014–2017年深圳地区侵袭性真菌病的菌种分类及分布情况，发现深圳地区侵袭性真菌感染的主要病原菌为念珠菌，尤以白色念珠菌为甚，其次是马尔尼菲青霉菌和曲霉菌，其临床感染主要出现在呼吸系统和消化系统。白色念珠菌、马尔尼菲青霉菌、热带念珠菌和真菌总数在2014–2016前三年逐渐增高，到2017年时转而下降；而其他真菌年度分离数量并没有明显的变化趋势。目前已发表研究性论文3篇，其中SCI论文2篇、中文期刊论文1篇；培养在读硕士研究生、副高级副主任技师和生物技术研究实习员各1名；初步建立基于RPA-LFD技术的隐球菌性脑膜炎、结核性脑膜炎鉴别诊断方法并评估其临床诊断价值，能快速、可视化检测新型隐球菌、格特隐球菌与结核分枝杆菌，为研发相关诊断试剂或设备提供了坚实的实验基础，为其他感染性疾病的可视化诊断提供了新的技术手段。