中华检验医学杂志
中華檢驗醫學雜誌
중화검험의학잡지
CHINESE JOURNAL OF LABORATORY MEDICINE
2008年
4期
429-432
,共4页
孙桂芝%高铁杰%钟镐镐%康丽军%张治国%衡万杰%吴秉铨%刘威
孫桂芝%高鐵傑%鐘鎬鎬%康麗軍%張治國%衡萬傑%吳秉銓%劉威
손계지%고철걸%종호호%강려군%장치국%형만걸%오병전%류위
聚合酶链反应%分枝杆菌,结核%细菌蛋白质类%突变%利福平%抗药性,细菌
聚閤酶鏈反應%分枝桿菌,結覈%細菌蛋白質類%突變%利福平%抗藥性,細菌
취합매련반응%분지간균,결핵%세균단백질류%돌변%리복평%항약성,세균
Polymerase chain reaction%Mycobactsrium tuberculosis%Bacterial proteins%Mutation%Rifampin%Drug resistance,bactenial
目的 应用实时荧光PCR分子信标技术,建立快速检测临床标本中结核分枝杆菌利福平rpoB相关耐药突变点方法,探讨其缩短耐药实验报告时间的临床应用价值.方法 以分枝杆菌药物敏感性实验绝对浓度法为标准,12株非结核分枝杆菌、4株非分枝杆菌作对照,对174例结核患者临床分离株应用实时荧光PCR分子信标方法,检测利福平rpoB核心区域的耐药突变点并将结果与直接测序进行比较.结果 (1)实时荧光PCR分子信标方法:82例结核分枝杆菌利福平敏感菌株中,3例发生rpoB基因突变,特异度为96.3%;92例结核分枝杆菌利福平耐药菌株中,82例检出耐药突变,敏感度为89.1%;准确性为92.5%.(2)DNA直接测序分析:82例结核分枝杆菌利福平敏感株中,1例发生rpoB基因突变,特异度为98.8%;92例结核分枝杆菌利福平耐药菌株中,83例发生:rpoB基因突变,敏感度为90.2%;准确性为94.2%.检测174株结核分枝杆菌临床分离菌株,与实时荧光PCR分子信标方法检测一致性为98.3%(171/174).结论 实时荧光PCR分子信标方法检测耐利福平结核分枝杆菌rpoB基因突变点可作为结核患者快速耐药检测的初筛方法之一.
目的 應用實時熒光PCR分子信標技術,建立快速檢測臨床標本中結覈分枝桿菌利福平rpoB相關耐藥突變點方法,探討其縮短耐藥實驗報告時間的臨床應用價值.方法 以分枝桿菌藥物敏感性實驗絕對濃度法為標準,12株非結覈分枝桿菌、4株非分枝桿菌作對照,對174例結覈患者臨床分離株應用實時熒光PCR分子信標方法,檢測利福平rpoB覈心區域的耐藥突變點併將結果與直接測序進行比較.結果 (1)實時熒光PCR分子信標方法:82例結覈分枝桿菌利福平敏感菌株中,3例髮生rpoB基因突變,特異度為96.3%;92例結覈分枝桿菌利福平耐藥菌株中,82例檢齣耐藥突變,敏感度為89.1%;準確性為92.5%.(2)DNA直接測序分析:82例結覈分枝桿菌利福平敏感株中,1例髮生rpoB基因突變,特異度為98.8%;92例結覈分枝桿菌利福平耐藥菌株中,83例髮生:rpoB基因突變,敏感度為90.2%;準確性為94.2%.檢測174株結覈分枝桿菌臨床分離菌株,與實時熒光PCR分子信標方法檢測一緻性為98.3%(171/174).結論 實時熒光PCR分子信標方法檢測耐利福平結覈分枝桿菌rpoB基因突變點可作為結覈患者快速耐藥檢測的初篩方法之一.
목적 응용실시형광PCR분자신표기술,건립쾌속검측림상표본중결핵분지간균리복평rpoB상관내약돌변점방법,탐토기축단내약실험보고시간적림상응용개치.방법 이분지간균약물민감성실험절대농도법위표준,12주비결핵분지간균、4주비분지간균작대조,대174례결핵환자림상분리주응용실시형광PCR분자신표방법,검측리복평rpoB핵심구역적내약돌변점병장결과여직접측서진행비교.결과 (1)실시형광PCR분자신표방법:82례결핵분지간균리복평민감균주중,3례발생rpoB기인돌변,특이도위96.3%;92례결핵분지간균리복평내약균주중,82례검출내약돌변,민감도위89.1%;준학성위92.5%.(2)DNA직접측서분석:82례결핵분지간균리복평민감주중,1례발생rpoB기인돌변,특이도위98.8%;92례결핵분지간균리복평내약균주중,83례발생:rpoB기인돌변,민감도위90.2%;준학성위94.2%.검측174주결핵분지간균림상분리균주,여실시형광PCR분자신표방법검측일치성위98.3%(171/174).결론 실시형광PCR분자신표방법검측내리복평결핵분지간균rpoB기인돌변점가작위결핵환자쾌속내약검측적초사방법지일.
Objective To establish a rapid method to detect mutations in rpoB genes of rifampin-resistant Mycobacterium tubereulosis in dinical specimens using Real-time fluorescence PCR molecular beacon assay.Methods 174 strains of Mvcobacterium tuberculosis clinical isolates were analyzed using real-time fluorescence PCR molecular beacon assay foilowed with DNA sequencing while 12 strains of NTM and 4 strains of bacteria other than Mycobacterium tuberculosis were used as the contrast.Results Eighty-two 89.1 of 92 rifampin (RIF)-resistant strains and 3 of 82 RIF-sensitive strains were found to harbor mutation in the rpoB gene using real-time fluorescence PCR-molecular beacon assay.The specificity, sensitivity,and accuracy of this assay were 96.3%,89.1%,and 92.5%,respectively-Eithty-three of 92 RIF-resistant strains and 1 of 82 RIF-sensitive strains were found to harbor mutation in the rpoB gene using the direct DNA sequencing.The specificity,sensitivity,and accuracy of the direct DNA sequencing were 98.8,90.2%,and 94.2%,respectively.As compared with real-time PCR molecular beacon assay,171 of 174(98.3%)strains of myeobactefium tuberculosis clinical isolates had the salne results.Conclusion Real-time fluorescence PCR-molecular beacon assay can be used as a rapid screen method to detect RIF-resistant isolates.
