实时定量PCR检测人全血标本中烟曲霉基因组载量的方法及临床应用

目的建立实时定量PCR（RQ-PCR）快速检测人全血标本中烟曲霉基因组载量的方法及进行初步临床应用。方法基于烟曲霉多拷贝基因ITSl-5．8S基因设计引物和TaqMan探针，用QIAamp^DNA Blood Mini Kit提取烟曲霉基因组DNA，建立20μlRQ-PCR反应体系，对含有不同载量烟曲霉基因组的模拟人全血标本和66份外科发热患者全血标本进行烟曲霉基因组的定量检测。结果检测限为10^-1基因组／μl上机待测液（即约78CFU／ml全血）；检测特异度和灵敏度分别为94．25％和99．04％，阳性预告值和阴性预告值分别为97．63％和97．62％；测定结果的平均相对误差为（3．67±13．19）％；批内及批间平均重复性变异系数分别为（12．38±1．53）％和（16．27±2．72）％；人血标本中烟曲霉基因组平均回收率为（107．81±25．92）％，回收率平均变异系数为（26．24±5．62）％。66份外科发热患者血标本中未检测出烟曲霉基因组。结论RQ-PCR可以快速、特异、灵敏地定量检测人血标本中烟曲霉基因组的载量，且有着较好的准确度与精密度。本研究外科发热患者血中未检测到烟曲霉基因组。

Detection of Aspergillus fumigatus genome load in human whole blood samples by real-time quantitative poly- merase chain reaction and its clinical application

Objective To establish a real-time quantitative polymerase chain reaction （RQ-PCR） assay for fast detection ofAspergillusfumigatus genome in human whole blood samples and explore its clinical application. Methods The primers and the TaqMan-probe were designed on the basis of the muhi-copy ITS1-5.8S region of the rDNA of Aspergillus fumigatus. The Aspergillus fumigatus genomic DNA were extracted with QIAamp~ DNA Blood Mini Kit. A 20 μl RQ-PCR amplification system was established, and the simulated blood samples containing various given load of Aspergillus fumigatus genome and the 66 whole blood samples of the surgical febrile patients were examined. Results The detection limit of the RQ-PCR instrument is 10^ -1 genomes/μl DNA sample, namely 78 CFU/ml whole blood. The specificity and the sensitivity were 94.25% and 99.04% respectively; and the positive predictive value and negative predictive value were 97.63% and 97.62% respectively. The average relative error of the quantitative results was （3.67 ± 13.19）%, and the intra- and the inter-assay average coefficients of variation were （ 12. 38 ± 1.53 ） % and （ 16. 27 ± 2. 72） %, respectively. The average recovery rate of Aspergillusfumigatus genomic DNA in human whole blood samples was （107.81 _＋ 25.92）%, and the average coeffi- cient of variation of the average recovery rate was （ 26. 24 ± 5.62 ） % . No Aspergillus fumigatus genomic DNA was detected among the 66 blood samples of the surgical febrile patients. Conclusions The RQ-PCR assay for fast quantitative detection of Aspergillus fumigatus genome in human whole blood samples is of high sensitivity, specificity,accuracy and precision. The AspergiLLus fumigatus genome was not detected in this group of surgical febrile patients.