中华临床营养杂志
中華臨床營養雜誌
중화림상영양잡지
CHINESE JOURNAL OF CLINICAL NUTRITION
2012年
4期
229-233
,共5页
徐云飞%马恩陵%康军仁%崔希增
徐雲飛%馬恩陵%康軍仁%崔希增
서운비%마은릉%강군인%최희증
肠外营养%实时定量PCR%通用真菌引物和探针%肠黏膜屏障%外科感染
腸外營養%實時定量PCR%通用真菌引物和探針%腸黏膜屏障%外科感染
장외영양%실시정량PCR%통용진균인물화탐침%장점막병장%외과감염
Parenteral nutrition%Real-time quantitative PCR%Universal fungi primers and probe%Gut barrier%Surgical infection
目的 建立实时定量PCR(RQ-PCR)以真菌通用引物和探针快速准确检测人全血标本中侵袭性真菌DNA载量的方法,并与细菌相鉴别及进行初步临床应用.方法 选择临床常见的真菌基因组多拷贝基因5.8S rDNA作为靶基因设计特异性通用真菌引物和TaqMan探针,采用QIAamp(R)血液DNA小提试剂盒提取多种致病真菌基因组DNA,建立20μl RQ-PCR反应体系,对含有不同载量致病真菌的模拟人全血标本和71份外科发热患者全血标本进行侵袭性真菌基因组的定量检测.结果 本方法的特异性良好,检测限为101拷贝/μl上机待测液(即约105拷贝/ml全血);检测灵敏度和特异度分别为95.5％和97.6％,阳性预告值和阴性预告值分别为98.7％和92.0％;标准曲线R2在0.9931～ 0.9977;批内及批间平均变异系数分别为(10.4±4.0)％和(27.9±2.0)％;人血标本中真菌基因组DNA平均回收率为(91.0±7.6)％,相对回收率平均变异系数为(14.9±4.0)％.71份外科发热患者血标本中未检测出侵袭性真菌基因组.结论 RQ-PCR可以借通用真菌引物和TaqMan探针快速、特异、灵敏地定量检测人血标本中侵袭性真菌DNA的载量并可与细菌相鉴别,且有着较好的准确度与精密度.外科发热患者血中侵袭性真菌基因组的存在率可能很低.
目的 建立實時定量PCR(RQ-PCR)以真菌通用引物和探針快速準確檢測人全血標本中侵襲性真菌DNA載量的方法,併與細菌相鑒彆及進行初步臨床應用.方法 選擇臨床常見的真菌基因組多拷貝基因5.8S rDNA作為靶基因設計特異性通用真菌引物和TaqMan探針,採用QIAamp(R)血液DNA小提試劑盒提取多種緻病真菌基因組DNA,建立20μl RQ-PCR反應體繫,對含有不同載量緻病真菌的模擬人全血標本和71份外科髮熱患者全血標本進行侵襲性真菌基因組的定量檢測.結果 本方法的特異性良好,檢測限為101拷貝/μl上機待測液(即約105拷貝/ml全血);檢測靈敏度和特異度分彆為95.5％和97.6％,暘性預告值和陰性預告值分彆為98.7％和92.0％;標準麯線R2在0.9931～ 0.9977;批內及批間平均變異繫數分彆為(10.4±4.0)％和(27.9±2.0)％;人血標本中真菌基因組DNA平均迴收率為(91.0±7.6)％,相對迴收率平均變異繫數為(14.9±4.0)％.71份外科髮熱患者血標本中未檢測齣侵襲性真菌基因組.結論 RQ-PCR可以藉通用真菌引物和TaqMan探針快速、特異、靈敏地定量檢測人血標本中侵襲性真菌DNA的載量併可與細菌相鑒彆,且有著較好的準確度與精密度.外科髮熱患者血中侵襲性真菌基因組的存在率可能很低.
목적 건립실시정량PCR(RQ-PCR)이진균통용인물화탐침쾌속준학검측인전혈표본중침습성진균DNA재량적방법,병여세균상감별급진행초보림상응용.방법 선택림상상견적진균기인조다고패기인5.8S rDNA작위파기인설계특이성통용진균인물화TaqMan탐침,채용QIAamp(R)혈액DNA소제시제합제취다충치병진균기인조DNA,건립20μl RQ-PCR반응체계,대함유불동재량치병진균적모의인전혈표본화71빈외과발열환자전혈표본진행침습성진균기인조적정량검측.결과 본방법적특이성량호,검측한위101고패/μl상궤대측액(즉약105고패/ml전혈);검측령민도화특이도분별위95.5％화97.6％,양성예고치화음성예고치분별위98.7％화92.0％;표준곡선R2재0.9931～ 0.9977;비내급비간평균변이계수분별위(10.4±4.0)％화(27.9±2.0)％;인혈표본중진균기인조DNA평균회수솔위(91.0±7.6)％,상대회수솔평균변이계수위(14.9±4.0)％.71빈외과발열환자혈표본중미검측출침습성진균기인조.결론 RQ-PCR가이차통용진균인물화TaqMan탐침쾌속、특이、령민지정량검측인혈표본중침습성진균DNA적재량병가여세균상감별,차유착교호적준학도여정밀도.외과발열환자혈중침습성진균기인조적존재솔가능흔저.
Objective To establish a real-time quantitative PCR(RQ-PCR)assay for fast detection of invasive fungi DNA in human whole blood samples with universal fungi primers and probe.Methods The universal fungi primers and the TaqMan-probe were designed on the basis of the multi-copy 5.8S region of the rDNA of the clinically most common invasive fungi.The invasive fungi 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 invasive fungi genome and the 71 whole blood samples of the surgical febrile patients were examined.Results The detection limit is 101 copies/μl amplification mixture,namely 105 copies/ml whole blood.The sensitivity and the specificity were 95.5％ and 97.6％,respectively; and the positive predictive value and negative predictive value were 98.7％ and 92.0％,respectively.The correlation coefficient of standard curve was between 0.9931 and 0.9977.The intra-and the inter-assay average coefficients of variation were(10.4 ±4.0)％ and(27.9 ± 2.0)％,respectively.The average relative recovery rate of fungi genomic DNA in blood samples was(91.0 ±7.6)％,and the average coefficients of variation of the relative recovery rate was(14.9 ±4.0)％.No fungi DNA was detected among the 71 blood samples of the surgical febrile patients.Conclusions The RQ-PCR assay for fast quantitative detection of invasive fungal DNA in human whole blood samples with the universal fungi primers and the TaqMan-probe was of high sensitivity,specificity,accuracy and precision,and is able to discriminate fungi from bacteria.The invasive fungi genome was not detected in this group of surgical patients,which may imply the less possibility of fungi translocation in the surgical febrile patients.
