CAJ | 학술논문

目的建立一种适合基层检验部门及小型实验室使用的快速检测嗜肺军团菌的方法.方法应用环介导等温扩增技术(LAMP),针对嗜肺军团菌mip基因设计5条引物(2条内引物、2条外引物及1条环引物),并对LAMP反应条件和反应体系进行优化.为验证该方法的特异性、敏感性,针对种系背景明确的12株嗜肺军团菌实验对照菌株、45株嗜肺军团菌地方分离株、6株非嗜肺军团菌、11株其他细菌以及59份外环境水样本进行检测,并将其结果与传统培养分离方法及定量PCR方法比较.结果所检测的菌株样本中不同血清型嗜肺军团菌经LAMP检测均呈绿色为阳性,非嗜肺军团菌及其他菌株检测均呈橙色为阴性.实验结果表明,LAMP方法的检出率高于传统培养分离方法及定量PCR.应用该方法从菌株核酸的提取至检测完成仅需1.5 h左右,该体系检测灵敏度为5 cfu/ml,而且其检测结果在日(灯)光下通过肉眼即可判断.结论实验建立的LAMP方法能够快速、灵敏、特异地检测嗜肺军团菌,适合基层检验部门及小型实验室与现场监测等使用.
목적 건립일충괄합기층검험부문급소형실험실사용적쾌속검측기폐군단균적방법.방법 응용배개도등온확증기술(LAMP),침대기폐군단균mip기인설계5조인물(2조내인물、2조외인물급1조배인물),병대LAMP반응조건화반응체계진행우화.위험증해방법적특이성、민감성,침대충계배경명학적12주기폐군단균실험대조균주、45주기폐군단균지방분리주、6주비기폐군단균、11주기타세균이급59빈외배경수양본진행검측,병장기결과여전통배양분리방법급정량PCR방법비교.결과 소검측적균주양본중불동혈청형기폐군단균경LAMP검측균정록색위양성,비기폐군단균급기타균주검측균정등색위음성.실험결과표명,LAMP방법적검출솔고우전통배양분리방법급정량PCR.응용해방법종균주핵산적제취지검측완성부수1.5 h좌우,해체계검측령민도위5 cfu/ml,이차기검측결과재일(등)광하통과육안즉가판단.결론 실험건립적LAMP방법능구쾌속、령민、특이지검측기폐군단균,괄합기층검험부문급소형실험실여현장감측등사용.
Objective To develop a loop-mediated isothermal amplification (LAMP) method for rapid diagnosing of Legionella pneumophila in the Pathogen Detection Department(PDD) or in small-scale laboratory. Methods Five primers (2 Inner Primers, 2 Outer Primers and a Loop Primer) for the LAMP test were designed by targeting the mip gene of Lpneumophila and reaction system of LAMP reaction was optimized. 12 strains of L.pneumophila, 45 local strains, 6 non-L.pneumophila strains, 11 other strains and 59 environmental water samples were analyzed to evaluate the specificity and sensibility of the LAMP amplification. At the same time, the results of the LAMP were also compared with biochemical culture and quantitative PCR methods. Results The amplification products of L.pneumophila turned green by visual inspection and had ladder-like pattern on the gel, but non-L.pneumophila and other products from the strains remained orange by visual examination and had no band on the gel. The detection rate of LAMP was higher than the biochemical culture and the real-time PCR methods. Reaction time of the LAMP method was only 1.5 h and the detection limit of LAMP assay was 5 cfu/reaction. In addition, the LAMP results could be determined only by visual inspection. Conclusion LAMP assay targeting the mip gene of L.pneumophila appeared to be rapid, specific, and sensitive for the detection of L.pneumophila. This method not only reduced the dependence of complicated equipment but also had a potential for wider use in the PDD, small-scale laboratory, emergency motor vehicle or for field survey.