CAJ | 학술논문

目的利用实时定量PCR技术(SYBR Green染料法)建立一种快速、特异、敏感、经济、准确检测肺炎支原体的方法,并评价其在临床上的应用价值.方法选择肺炎支原体的23SrRNA耐药相关基因作为扩增区,设计特异性引物,然后提取相应质粒作为标准品,建立绝对定量的标准曲线.比较荧光定量PCR方法与传统巢式PCR方法和市售试剂盒的特异度和灵敏度;分别利用上述三种方法和培养法检测临床咽拭子样本,进行相关系数、符合率及kappa系数的分析.结果实时定量PCR和巢式PCR检测肺炎支原体的灵敏度为10拷贝;市售试剂盒为102拷贝.特异性检验中,肺炎支原体样本检测为阳性,人型支原体和其他4种细菌均检测为阴性.临床样本检测中,实时定量PCR、巢式PCR、市售试剂盒和培养法的阳性率分别为55.49％、52.75％、47.25％和39.01％.统计学分析结果显示,新建实时定量PCR与传统巢式PCR的结果总符合率为89.6％,Kappa系数为0.790.实时定量PCR方法与市售试剂盒检测结果的相关系数R=0.923,P＜0.001;两种方法的总符合率为89.6％,Kappa系数为0.792.实时定量PCR与培养法的结果总符合率为83.5％,Kappa系数为0.678.结论采用实时定量PCR方法检测肺炎支原体与其它几种方法相比,快速经济,且污染少,特异性强,灵敏度高,具有很重要的科研和临床实用价值.
목적 이용실시정량PCR기술(SYBR Green염료법)건립일충쾌속、특이、민감、경제、준학검측폐염지원체적방법,병평개기재림상상적응용개치.방법 선택폐염지원체적23SrRNA내약상관기인작위확증구,설계특이성인물,연후제취상응질립작위표준품,건립절대정량적표준곡선.비교형광정량PCR방법여전통소식PCR방법화시수시제합적특이도화령민도;분별이용상술삼충방법화배양법검측림상인식자양본,진행상관계수、부합솔급kappa계수적분석.결과 실시정량PCR화소식PCR검측폐염지원체적령민도위10고패;시수시제합위102고패.특이성검험중,폐염지원체양본검측위양성,인형지원체화기타4충세균균검측위음성.림상양본검측중,실시정량PCR、소식PCR、시수시제합화배양법적양성솔분별위55.49％、52.75％、47.25％화39.01％.통계학분석결과현시,신건실시정량PCR여전통소식PCR적결과총부합솔위89.6％,Kappa계수위0.790.실시정량PCR방법여시수시제합검측결과적상관계수R=0.923,P＜0.001;량충방법적총부합솔위89.6％,Kappa계수위0.792.실시정량PCR여배양법적결과총부합솔위83.5％,Kappa계수위0.678.결론 채용실시정량PCR방법검측폐염지원체여기타궤충방법상비,쾌속경제,차오염소,특이성강,령민도고,구유흔중요적과연화림상실용개치.
Objective To develope a new Real-time quantitative PCR assay using SYBR green as fluorescence reporter, which is rapid, specific, sensitive, cheap and accurate for the detection of mycoplasma pneumoniae(MP), and evaluated its clinical application value.Methods The sequence of the 23S rRNA gene in MP type strain FH was selected as amplified regions, and specific primers were designed.Then the related plasmids were extracted as standards,and the absolute quantitative standard curve was established.The sensitivity ,specificity of the fluorescence quantitative PCR assay was compared with the nest-PCR and kit;To calculate correlation coefficient, coincidence rate and kappa coefficient, clinical samples were detected using above-mentioned methods and cultivation,respectively.Results The detection sensitivity of the new real-time PCR and nest-PCR was 10 copies of FH DNA,while the kit 100 copies.In the specificity tests,the MP sample was positive,while mycoplasma hominis and other four bacteria were all negative.We applied this real-time PCR assay ,nest-PCR, kit and cultivation to 182 clinical specimens, and the detection rates were 55.49％, 52.75％, 47.25％ and 39.01％ ,respectively.The total consistency rate and Kappa coefficient of the new real-time PCR method and nest-PCR were 89.6％ ,0.790, respectively;while those of the new method and cultivation were 83.5 ％ ,0.678, respectively.The total consistency rate and Kappa coefficient of the new real-time PCR method and the kit were 89.6％ ,0.792,respectively;and the correlation coefficient of these two methods was 0.923,P ＜ 0.001.Conclusion Compared with other methods, the new real-time PCR assay could be used to detect mycoplasma pneumoniae quickly and economically, with high sensitivity and specificity ,revealing great utility value on varied instrumentation platforms.