基于重组甘露糖结合凝集素蛋白–磁珠富集技术检测血流感染新型隐球菌的方法  

Detection assay of Cryptococcus neoformans bloodstream infection based on recombined MBL-magnetic beads enrichment

作　　者：陈晓丽 李文革[1] 郑皓 陈小萍[1] 卢金星[1] Chen Xiaoli;Li Wenge;Zheng Hao;Chen Xiaoping;Lu Jinxing(State Key Laboratory of Communicable Disease Prevention and Control,National Institute for Communicable Disease Prevention and Control,Chinese Center for Disease Control and Prevention,Beijing 102206,China)

机构地区：[1]中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室,北京102206

出　　处：《疾病监测》2020年第10期939-945,共7页Disease Surveillance

基　　金：国家科技重大专项(No.2018ZX10733–402)。

摘　　要：目的建立一种基于重组甘露糖结合凝集素(MBL)蛋白–磁珠富集技术快速检测血流感染新型隐球菌的方法,为临床确诊新型隐球菌感染提供指导。方法利用Western blot方法检测重组MBL蛋白(M1)与新型隐球菌定性结合的情况;利用磷酸缓冲盐溶液(PBS)与兔血模拟样本检测M1-protein A磁珠对不同浓度梯度的新型隐球菌的捕获情况;比较3种不同方法[方法1,M1-protein A磁珠富集后直接进行qPCR鉴定;方法 2,M1-protein A磁珠富集后培养加质谱鉴定;方法 3(金标准),血培养加质谱鉴定]检测血液新型隐球菌的优劣。结果 M1蛋白与新型隐球菌标准株及临床株普遍结合;不同浓度梯度新型隐球菌的PBS与兔血模拟阳性样本中,M1-protein A磁珠复合体的富集效率为18.00%~83.00%;3种新型隐球菌鉴定方法中,方法1耗时最短(4.25 h),灵敏度为10菌落形成单位/毫升(CFU/ml);方法 2灵敏度可达到≤1 CFU/ml,总时间57 h;作为临床诊断金标准的方法 3灵敏度虽也能达到≤1 CFU/ml,但耗时较长,需120 h。结论采用磁珠富集结合qPCR、MALDI-TOF MS技术,比传统血培养大大缩短了检测时间,并且具有较高的灵敏度和特异度。Objective To establish an assay based on recombined MBL protein-magnetic beads enrichment technique for the detection of Cryptococcus neoformans directly from whole blood, and provide guidance for clinical diagnosis of C. neoformans infection. Methods Western blot was used to detect the direct binding of recombined MBL protein(M1) to C. neoformans. M1-protein A beads were used to enrich C. neoformans with different concentration gradients in phosphate buffer saline(PBS)-simulated and rabbit blood-simulated samples, and the enrichment efficiencies were obtained. Three different assays of detecting C. neoformans in rabbit blood-simulated samples were compared., i.e. assay 1, M1-protein A enrichment followed by qPCR identification, assay 2, M1-protein A enrichment followed by culture and MS identification,assay 3, blood culture followed by MS identification(standard gold method). Results M1 protein could bind with standard and clinical strains of C. neoformans. The enrichment efficiency of M1-protein A magnetic beads in PBS-simulated samples with pathogens of 20 CFU/ml, 10 CFU/ml, 5 CFU/ml, and 1 CFU/ml was 71.65%, 72.40%, 62.70%, and 83.00% respectively,and in rabbit blood-simulated samples was 35.76%, 18.00%, 21.04%, and 23.30% respectively. Among the three assays of detecting C. neoformans, assay 1 needed the shortest time(4.25 h), and sensitivity was 10 CFU/ml. The sensitivity of assay 2 could reach ≤ 1 CFU/ml and needed a total time of 57 h. Although the sensitivity of assay 3 could also reach ≤ 1 CFU/ml, it lasted for 120 h. Conclusion Compared with standard blood culture, the detection assays with qPCR or MALDI-TOF MS based on M1-protein A beads enrichment need much less time, but still have high sensitivity and specificity.

关 键 词：甘露糖结合凝集素 磁珠富集 血流感染 新型隐球菌 

分 类 号：R211[医药卫生—中医学] R379.5