多重实时定量PCR同时检测人全血中大肠杆菌与白念珠菌基因方法的建立  

Multiplex real-time quantitative polymerase chain reaction for simultaneous detection of Escherichia coli and Candida albicans genes in human whole blood

作　　者：房嘉宾[1] 康军仁[1] 马恩陵[1] 郭广亮[1] 崔希增[1] 

机构地区：[1]中国医学科学院北京协和医学院北京协和医院肠外肠内营养科,100730

出　　处：《中华临床营养杂志》2015年第3期170-175,共6页Chinese Journal of Clinical Nutrition

基　　金：北京市联合攻关项目基金（2002-1024）;卫生部临床学科重点项目基金（20010103）

摘　　要：目的建立多重实时定量PCR（MRQ—PCR）同时快速检测血中大肠杆菌与白念珠菌DNA的方法，以评估肠屏障损伤可能导致的移位微生物的种类和程度并提供针对性用药的建议。方法选择大肠杆菌β-右旋半乳糖苷酶基因作为检测大肠杆菌的靶基因设计引物和探针，选择白念珠菌ITS2基因设计引物和探针。采用QIAamp DNA Blood Mini Kit试剂盒提取大肠杆菌与白念珠菌基因组DNA；建立25μlTaqMan探针MRQ-PCR反应体系；对18份模拟血标本及10份外科发热患者临床标本进行MRQ—PCR检测。结果引物和探针特异性好，大肠杆菌与白念珠菌标准曲线相关系数分别在0．994—0．999和0．994～0．998，扩增效率分别为0．894～1．022和0．905～1．028。标准样品检测限分别为大肠杆菌13．9拷贝／μl和白念珠菌0．8cfu／μl，两种菌的检测灵敏度分别为100％和99．69％，特异度分别为100％和94．73％，标准品中大肠杆菌与白念珠菌特异基因平均回收率分别为（101．89±5．69）％和（103．74±4．64）％，两种菌基因检测的批内变异系数分别为（13．14±10．27）％和（19．18±8．54）％，批间变异系数分别为（14．35±9．34）％和（18．31±10．25）％。全血标本中大肠杆菌与白念珠菌特异基因的检出限分别为12455．2拷贝／ml和800．3cfu／ml，平均回收率分别为（111．60±11．06）％和（99．96±6．16）％，两种菌基因检测的批内变异系数分别为（11．02±5．65）％和（8．14±7．29）％，平均批间变异系数分别为（12．88±7．59）％和（18．62±9．14）％。结论多重实时定量PCR可以同时快速、灵敏、特异地定量检测人全血标本中大肠杆菌与白念珠菌基因，准确度高、重复性好、节省血标本用量及检测成本、总检测时间缩短，在临床全血标本的真菌与细菌快速鉴别检测、抗微生物药物的针对性应用及疗效评估、肠屏障�Objective To establish a multiplex real-time quantitative polymerase chain reaction （ MRQPCR） assay for fast and simultaneous detection of Escheriehia coli （E. coli） and Candida albicans （C. albicans） genes in human whole blood, in order to facilitate differentiation of the types of microorganism and evaluation of the severity of bacterial or fungi translocation due to impaired gut barrier, hence providing help to select specific antimicrobial agents. Methods The β-D-galactosidase gene of E. coli and ITS2 gene of C. albicans were selected as the target genes for designing primers and probes. E. coli and C. albicans genomes were extracted with QIAamp DNA Blood Mini Kit, and the 25 μl TaqMan MRQ-PCR amplification reaction system was established. 18 simulated human whole blood samples and 10 whole blood samples from febrile surgical patients were detected for E. coli and C. albicans genes using MRQ-PCR. Results The specificity of the primers and probes were excellent. The correlation coefficients of the standard curves of E. coli and C. albicans were 0. 994 - 0. 999 and 0. 994 - 0. 998, respectively ; and the efficiency of amplification were 0. 894 - 1. 022 and 0. 905 - 1. 028, respectively. In the standard samples, the lowest detection limits of E. coli and C. albicans were 13.9 copies/μl and 0. 8 cfu/μl, respectively; the sensitivity was 100% and 99. 69%, the specificity was 100% and 94.73%, respectively; the average recovery rates were （101.89 ± 5.69）% and （103.74 ± 4. 64）% respectively; the intra-batch coefficients of variance （CV） in detecting the genes were （13. 14 ± 10. 27） % and （ 19. 18 ± 8. 54） %, respectively, and the inter-batch CV were （ 14. 35 ± 9. 34） % and （ 18. 31 ± 10. 25 ） % , respectively. In human whole blood, the lowest detection limits of E. coli and C. albicans were 12 455.2 copies/ml and 800. 3 cfu/ml, respectively; the average recovery rates were （ 111.60 ± 11.06）% and （ 99. 96 ± 6. 16） %, respectively; the intra-batch CV in detect

关 键 词：多重实时定量PCR 大肠杆菌 白念珠菌 全血 

分 类 号：R446.5[医药卫生—诊断学]