中华临床营养杂志
中華臨床營養雜誌
중화림상영양잡지
CHINESE JOURNAL OF CLINICAL NUTRITION
2011年
3期
154-158
,共5页
崔希增%马恩陵%康军仁%郭广亮%房嘉宾%徐云飞
崔希增%馬恩陵%康軍仁%郭廣亮%房嘉賓%徐雲飛
최희증%마은릉%강군인%곽엄량%방가빈%서운비
实时定量PCR%烟曲霉%全血%外科感染
實時定量PCR%煙麯黴%全血%外科感染
실시정량PCR%연곡매%전혈%외과감염
Real-time quantitative PCR%Aspergillus fumigalus%Human whole blood%Surgical infection
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ITS1-5.8S基因设计引物和TaqMan探针,用QIAamp(R)DNA Blood Mini Kit提取烟曲霉基因组DNA,建立20μl RQ-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78 CFU/ml全血);检测特异度和灵敏度分别为94.25%和99.04%,阳性预告值和阴件预告值分别为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.
目的 建立實時定量PCR(RQ-PCR)快速檢測人全血標本中煙麯黴基因組載量的方法及進行初步臨床應用.方法 基于煙麯黴多拷貝基因ITS1-5.8S基因設計引物和TaqMan探針,用QIAamp(R)DNA Blood Mini Kit提取煙麯黴基因組DNA,建立20μl RQ-PCR反應體繫,對含有不同載量煙麯黴基因組的模擬人全血標本和66份外科髮熱患者全血標本進行煙麯黴基因組的定量檢測.結果 檢測限為10-1基因組/μl上機待測液(即約78 CFU/ml全血);檢測特異度和靈敏度分彆為94.25%和99.04%,暘性預告值和陰件預告值分彆為97.63%和97.62%;測定結果的平均相對誤差為(3.67±13.19)%;批內及批間平均重複性變異繫數分彆為(12.38±1.53)%和(16.27±2.72)%;人血標本中煙麯黴基因組平均迴收率為(107.81±25.92)%,迴收率平均變異繫數為(26.24±5.62)%.66份外科髮熱患者血標本中未檢測齣煙麯黴基因組.結論 RQ-PCR可以快速、特異、靈敏地定量檢測人血標本中煙麯黴基因組的載量,且有著較好的準確度與精密度.本研究外科髮熱患者血中未檢測到煙麯黴基因組.
목적 건립실시정량PCR(RQ-PCR)쾌속검측인전혈표본중연곡매기인조재량적방법급진행초보림상응용.방법 기우연곡매다고패기인ITS1-5.8S기인설계인물화TaqMan탐침,용QIAamp(R)DNA Blood Mini Kit제취연곡매기인조DNA,건립20μl RQ-PCR반응체계,대함유불동재량연곡매기인조적모의인전혈표본화66빈외과발열환자전혈표본진행연곡매기인조적정량검측.결과 검측한위10-1기인조/μl상궤대측액(즉약78 CFU/ml전혈);검측특이도화령민도분별위94.25%화99.04%,양성예고치화음건예고치분별위97.63%화97.62%;측정결과적평균상대오차위(3.67±13.19)%;비내급비간평균중복성변이계수분별위(12.38±1.53)%화(16.27±2.72)%;인혈표본중연곡매기인조평균회수솔위(107.81±25.92)%,회수솔평균변이계수위(26.24±5.62)%.66빈외과발열환자혈표본중미검측출연곡매기인조.결론 RQ-PCR가이쾌속、특이、령민지정량검측인혈표본중연곡매기인조적재량,차유착교호적준학도여정밀도.본연구외과발열환자혈중미검측도연곡매기인조.
Objective To establish a real-time quantitative polymerase chain reaction (RQ-PCR) assay for fast detection of Aspergillus fumigatus 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 multi-copy ITS1-5. 8S region of the rDNA of Aspergillus fumigatus. The Aspergillus fumigatus genomic DNA were extracted with QIAamp(R) 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 Aspergillus fumigatus genomic DNA in human whole blood samples was (107. 81 ±25. 92)% , and the average coefficient 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.
