实时定量PCR快速检测外科发热患者血中白念珠菌载量的方法

目的 建立实时定量PCR(RQ-PCR)快速检测血中自念珠菌(Gandiad albicans)载量的方法,并对可能存在肠屏障损伤和肠道菌移位的外科发热患者标本进行检测.方法 基于白念珠菌基因组单拷贝基因NAG1设计引物和探针;构建并制备质粒做标准品;用QIAamp(R) DNA Blood Mini Kit试剂盒提取白念珠菌基因组DNA;建立20μl TaqMan探针RQ-PCR反应体系;对74份外科发热患者临床标本进行定量检测.结果 引物和探针特异性好;标准曲线R2 值0.9918～0.9985,扩增效率0.88～1.027,检测限为100 拷贝/μl上机检测液(即约1.1×103 cfu/ml全血),仪器检测灵敏度为97.46%,特异度为100%,平均准确性为(99.64±2.08)%,批内及批间重复性的平均变异系数分别为(14.76±2.64)%和(17.85±3.53)%;血标本中白念珠菌基因组平均提取效率为(88.60±5.73)%,提取效率平均变异系数为(11.70±5.36)%;标本于-20℃存放3、6个月后和0时间点定量结果比较,差异无显著性(P=0.267);74份外科发热患者血中白念珠菌的阳性率为2.7%,最高含量约为4.42×103 cfu/ml.结论 RQ-PCR可以快速、灵敏、特异地定量检测血标本中白念珠菌,重复性好,适用于临床日常检验.少数外科发热患者周围血中存在白念珠菌.

Detection of Candida albicans in the venous blood of the surgical febrile patients by real-time quantitative PCR

Objective To establish the real-time quantitative PCR (RQ-PCR) assay for detecting Candida albicans (C.albicans) in whole blood and its clinical application in the febrile surgical patients who may develop gut barrier damage and gut microorganism translocation.Methods The NAG1 gene,which is a single copy in C.albicans genome,was selected as the target gene for designing the primers and probe.The plasmid was fabricated and produced as standard samples.C.albicans genomes were extracted with QIAamp(R) DNA Blood Mini Kit,and the total 20 μl TaqMan RQ-PCR amplification reaction system was established.The 74 venous blood samples from the surgical febrile patients were detected for C.albicans load.Results The specificities of the primers and probe were excellent,the correlation coefficients of the standard curves were between 0.9918 and 0.9985,and the efficiency of amplification was 0.88-1.027 for the samples above the lowest detection limit (100 copies/μl examine fluid,or nearly 1.1 × 103 cfu/ml whole blood).The average accuracy of the RQ-PCR equipment was (99.64±2.08) %,the sensitivity was 97.46%,the specificity was 100%,and the average coefficients of variation (CV) of the intra-and inter-assay were (14.76±2.64)% and (17.85±3.53)%,respectively.The average recovery rate of C.albicans DNA in whole blood samples was (88.60±5.73) %,and the average CV of recovery rate was (11.70 ±5.36) %.The number of copies of C.albicans genes per unit blood was not significantly different among the same original blood samples stored separately under-20℃ for 3 or 6 months when compared with its freshly collected blood (P = 0.267).In the 74 whole blood samples obtained from the febrile surgical patients,the positive rate of C.albicans genes was 2.7% and the highest load was 4.42×103 cfu/ml.Conclusions RQ-PCR is a rapid,sensitive,highly specific,and reproducible method in detecting C.albicans NAG1 gene.Clinically it can be used to quantitatively evaluate the numbers of C.albicans in the whole blood.A small percentage of the febrile surgical patients may develop blood infection of C.albicans.