CAJ | 학술논문

目的环介导等温扩增(LAMP)技术检测卡氏肺孢子虫(Pc).方法醋酸可的松经皮下注射Wistar大鼠诱导Pc,收集支气管肺泡灌洗液(BALF)提取Pc基因组DNA.设计4条扩增Pc线粒体核糖体大亚基(mtrRNA)基因的LAMP引物,以结核杆菌、肺炎支原体、肺炎衣原体、弓形虫、大鼠白细胞为对照,进行LAMP反应.LAMP产物经显色、电泳及酶切鉴定.将Pc DNA 10倍稀释后同时进行LAMP和PCR,比较其敏感性.结果 Pc检测管经显色后呈绿色(阳性),对照组均呈棕色(阴性).Pc LAMP产物经电泳后呈LAMP特征性梯状条带,扩增产物经Tail限制性内切酶酶切鉴定正确,对照组均无扩增产物.LAMP可检测到虫体DNA的最低浓度是lP9/pJ,为PCR的10倍.结论检测Pc的LAMP方法敏感、特异及简便.
목적 배개도등온확증(LAMP)기술검측잡씨폐포자충(Pc).방법 작산가적송경피하주사Wistar대서유도Pc,수집지기관폐포관세액(BALF)제취Pc기인조DNA.설계4조확증Pc선립체핵당체대아기(mtrRNA)기인적LAMP인물,이결핵간균、폐염지원체、폐염의원체、궁형충、대서백세포위대조,진행LAMP반응.LAMP산물경현색、전영급매절감정.장Pc DNA 10배희석후동시진행LAMP화PCR,비교기민감성.결과 Pc검측관경현색후정록색(양성),대조조균정종색(음성).Pc LAMP산물경전영후정LAMP특정성제상조대,확증산물경Tail한제성내절매매절감정정학,대조조균무확증산물.LAMP가검측도충체DNA적최저농도시lP9/pJ,위PCR적10배.결론 검측Pc적LAMP방법민감、특이급간편.
Objective To detect Pneumocystis carinii (Pc) DNA by loop-mediated isothermal amplification (LAMP). Methods After injected with hydrocortisone acetate for 8 weeks, the bronchoalveolar lavage fluid (BALF) of Wistar rats were collected and a portion of BALF were examined for identifying Pc organisms using microscope. Then Pc DNA was extracted by phenol-chloroform extraction. Four primers which recognized 6 distinct regions on the mtrRNA gene of Pc were designed and used for LAMP assay. To evaluate the specificity of the assay, M. tuberculosis, M. pneumoniae, C. pneumoniae, P. gondii and rat leucocyte were used as negative controls. To compare the sensitivity of the LAMP to that of conventional PCR, Pc DNA were 10-fold serially diluted and was amplified by LAMP and PCR. LAMP results were judged by naked eye, electrophoretic analysis and restriction digestion. Results Pc organisms were detected from BALF of rats injected with hydrocortisone acetate. After LAMP reaction, positive signal was observef rats injected with hydrocortisone acetate. By contrast, no positive signal was observed for the negative controls in the study. The amplified product digested by restriction enzyme demonstrated 3 bands (82, 135, 189 lip) upon agarose gel electrophoresis, in good agreement with the predicted sizes. The detection limit of LAMP assay was 1 pg/μl of Pc DNA per reaction and that of PCR was 10 pg/μ1 of Pc DNA per reaction. Conclusion LAMP assay has usefulness for rapid detection of Pc.