CAJ | 학술논문

目的探讨ITS1-5.8S rRNA-ITS2巢式PCR法检测大鼠卡氏肺孢子虫的敏感性及早期检测的评估.方法采用地塞米松免疫抑制法诱导大鼠感染肺孢子虫,大鼠分为7组,6组实验组和1组对照组,每组10只,实验组采用地塞米松免疫抑制法诱导大鼠感染肺孢子虫,对照组则不予激素处理.自诱导开始第3周至第8周每周收集1组实验组大鼠肺组织(lung tissue,LT)和支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)标本,采用ITS1-5.8S rRNA-ITS2巢式PCR法扩增卡氏肺孢子虫ITS1-5.8S rRNA-ITS2,同时采用六亚甲基四胺银(GMS)染色镜检法对第3周到第8周的大鼠肺组织和肺泡灌洗液标本进行检测,并将两种方法进行比较以评估巢式PCR技术的敏感性.结果采用ITS1-5.8S rRNA-ITS2 巢式PCR 法对实验感染卡氏肺孢子虫的大鼠LT和BALF进行检测,卡氏肺孢子虫DNA阳性率依次为第3周20%(2/10)、0(0/10),第4周70%(7/10)、10%(1/10),第5周90%(9/10)、30%(3/10),第6周90%(9/10)、80%(8/10),第7周100%(10/10)、80%(8/10),第8周63%(5/8)、63%(5/8).而GMS染色镜检法仅于第5周开始检出卡氏肺孢子虫包囊,第6、7周包囊数最多.实验组大鼠诱导后的前4周无明显卡氏肺孢子虫肺炎体征,第5周后症状趋于明显.结论 ITS1-5.8S rRNA-ITS2巢式PCR法敏感性高,可在第3周后和无症状阶段实验大鼠中检出卡氏肺孢子虫,并且比镜检法早两周.
목적 탐토ITS1-5.8S rRNA-ITS2소식PCR법검측대서잡씨폐포자충적민감성급조기검측적평고.방법 채용지새미송면역억제법유도대서감염폐포자충,대서분위7조,6조실험조화1조대조조,매조10지,실험조채용지새미송면역억제법유도대서감염폐포자충,대조조칙불여격소처리.자유도개시제3주지제8주매주수집1조실험조대서폐조직(lung tissue,LT)화지기관폐포관세액(bronchoalveolar lavage fluid,BALF)표본,채용ITS1-5.8S rRNA-ITS2소식PCR법확증잡씨폐포자충ITS1-5.8S rRNA-ITS2,동시채용륙아갑기사알은(GMS)염색경검법대제3주도제8주적대서폐조직화폐포관세액표본진행검측,병장량충방법진행비교이평고소식PCR기술적민감성.결과 채용ITS1-5.8S rRNA-ITS2 소식PCR 법대실험감염잡씨폐포자충적대서LT화BALF진행검측,잡씨폐포자충DNA양성솔의차위제3주20%(2/10)、0(0/10),제4주70%(7/10)、10%(1/10),제5주90%(9/10)、30%(3/10),제6주90%(9/10)、80%(8/10),제7주100%(10/10)、80%(8/10),제8주63%(5/8)、63%(5/8).이GMS염색경검법부우제5주개시검출잡씨폐포자충포낭,제6、7주포낭수최다.실험조대서유도후적전4주무명현잡씨폐포자충폐염체정,제5주후증상추우명현.결론 ITS1-5.8S rRNA-ITS2소식PCR법민감성고,가재제3주후화무증상계단실험대서중검출잡씨폐포자충,병차비경검법조량주.
Objective To investigate the sensitivity and significance of nested PCR with ITS1-5.8S rRNA-ITS2 for early detection of Pneumocystis carinii (P. carinii) in rats. Methods Infection of P. carinii in rats was established by subcutaneous injection of an immunodepressant, dexamethasone. The study was carried out in 7 groups, 10 rats each, including one uninfected group as control. Samples of bronchoalveolar lavage fluid (BALF) and lung tissue (LT) from the rats were collected and assayed weekly after the 3 rd week of immunedepression by the nested PCR. The primer for ITS1-5.8S rRNA-ITS2 of P. carinii was used for the nested PCR. Meanwhile LT and BALF smears stained with Gomori(s) methenamine silver (GMS) were examined under microscope for P. carinii. Results Aspecific marker site of ITS1-5.8SrRNA-ITS2 of P. carinii from the samples of LT and BALF in the infected rats were amplified by the nested PCR, and it was found that the positive rates during 3-8 weeks were 20% and 0, 70% and 10%, 90% and 30%, 90% and 80%, 100% and 80% ,63% and 63%, respectively. In comparison, P. carinii was observed by the microscopic examination only at the 5th week post-infection, while the number of cysts reached to the highest at the 7th and 8th week.The experimental rats showed no obvious pneumonia symptoms before the 4th week, but becoming apparent at the 5th week and there after. Conclusion The nested PCR to identify specific marker site ITS1-5.8S rRNA-ITS2 is a sensitive method and could detect P. carinii3 weeks after infection while no obvious pneumonia symptoms in rats, 2 weeks earlier than the traditional microscopic method.