基于M1磁珠富集血液中念珠菌快速检测方法的建立  

Establishment of fast diagnostic assays of Candida spp.in blood samples based on M1 beads enrichment

作　　者：郑皓 李文革[1] 卢金星[1] 陈小萍[1] Zheng Hao;Li Wenge;Lu Jinxing;Chen Xiaoping(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,北京102206

出　　处：《疾病监测》2021年第6期622-627,共6页Disease Surveillance

基　　金：国家科技重大专项(No.2018ZX10733402)。

摘　　要：目的建立3种基于重组人甘露聚糖结合凝集素(MBL)蛋白(M1蛋白)磁珠富集技术的血液微量念珠菌快速检测方法(荧光定量PCR法、微培养法、铺板培养法)。方法根据荧光定量PCR方法的扩增效果,比较3种裂解液(酵母裂解液、LSTE和TXTE)提取微量念珠菌DNA的效果;通过检测液体培养后的菌量,比较5种微培养液(PBS、SA、YPD、LB和BHI)的增菌效果;使用人血浆模拟样本评价建立的3种方法和标准血培养法对血液中微量念珠菌的检测效果。结果与TXTE和LSTE相比,酵母裂解液提取微量念珠菌DNA的效果最佳。SA、YPD、LB和BHI 4种微培养液中的菌量随时间延长均有上升趋势,特别是BHI增菌效果最佳。人血浆模拟样本中,荧光定量PCR法虽然能够在最短时间内(3.75 h)检测到病原菌,但不能检出全部阳性样本。与传统血培养法相比,微培养法和铺板培养法可分别提前至少39 h和22 h获得检测结果,灵敏度为10菌落形成单位/mL(CFU/mL)。结论与传统血培养法相比,荧光定量PCR法、微培养法和铺板培养法均能有效缩短检测时间,并且有较高的特异性和灵敏度。Objective To establish three fast assays(real-time PCR,micro-culture and plate cultures assays)based on recombined mannose-binding lectin(MBL)protein-magnetic beads enrichment technique for the detection of Candida spp.directly from whole blood.Methods Three lysates(yeast lysate,LSTE and TXTE)were compared to determine the efficiency of Candida DNA extraction according to the amplification effects of real-time PCR.Five kinds of micro-culture medium(PBS,SA,YPD,LB and BHI)were compared for Candida culture efficiency by calculating the amount of Candida after culture.The efficiencies of three new assays for the Candida DNA extraction were evaluated using human plasma simulated samples and compared with standard blood cultures.Results Compared with TXTE and LSTE,yeast lysate had the best DNA extraction effect for trace amount of Candida.The amount of Candida in SA,YPD,LB and BHI micro-culture media increased with time,especially for BHI.In the human plasma simulated samples,real-time PCR assay could detect Candida in the shortest time(3.75 h),but it could not detect all positive samples.Compared with standard blood culture assay,the identification results of micro-culture and plate-culture were obtained at least 39 h and 22 h earlier,respectively,and the sensitivity could reach 10 CFU/mL.Conclusion Compared with standard blood culture assay,the real-time PCR,microculture and plate culture assays need much less time and have high specificity and sensitivity.

关 键 词：M1磁珠富集 念珠菌 念珠菌血症 检测方法 

分 类 号：R211[医药卫生—中医学] R379.4