摘要
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(1ungtissue,LT)和支气管肺泡灌洗液(bronchoal veolar lavage fluid,BALF)标本,采用ITSl-5.8SrRNA—ITS2巢式PCR法扩增卡氏肺孢子虫ITSI-5.8SrRNA—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ITSI-5,8SrRNA—ITS2巢式PCR法对实验感染卡氏肺孢子虫的大鼠¨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ITSI-5.8SrRNA-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。
Objective To investigate the sensitivity and significance of nested PCR with ITS1-5.8S rRNA-ITS2 for early detection of Pneumocystis carinii ( P. carinli) in rats. Methods Infection of P. carinii in rats was established by subcutaneous injection of an immunodepressant, dexamethasone. The study was car- ried out in 7 groups, i0 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 immunodepression 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 A specific marker site of ITS1-5.8S rRNA-ITS2 of P. carlnii 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-i^ffection, 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. carinii 3 weeks after infection while no obvious pneumonia symptoms in rats, 2 weeks earlier than the traditional microscopic method.
出处
《国际医学寄生虫病杂志》
CAS
2011年第1期10-13,共4页
International JOurnal of Medical Parasitic Diseases
基金
广西人事厅留学回国人员基金项目(桂人函2008第987号)
广西科技厅重点项目(重200951)
广西科技厅科技攻关项目(桂科攻0816004.39)