mtLSU-巢式PCR检测实验大鼠卡氏肺孢子虫的实验研究

目的对mtLSU-巢式PCR方法检测大鼠卡氏肺孢子虫的应用价值以及基因序列进行评价。方法采用地塞米松免疫抑制法诱导大鼠感染肺孢子虫;实验组10只,对照组1只;诱导至第7周时收集实验组及对照组大鼠肺组织和支气管肺泡灌洗液（BALF）标本,采用mtLSU-巢式PCR方法对人源与鼠源肺孢子虫共有的基因进行扩增和序列测定,同时采用镜检法对实验组大鼠肺组织和肺泡灌洗液标本进行检测,评估两种方法的敏感性。结果采用mtLSU-巢式PCR方法对实验感染大鼠肺组织和BAL进行检测,卡氏肺孢子虫DNA阳性率分别为100%（10/10）、90%（9/10）。而GMS染色镜检法检测的阳性率分别为80%（8/10）、60%（6/10）。所测Wistar大鼠卡氏肺孢子虫mtLSU基因序列长度为155bp,与GenBank的大鼠源肺孢子虫（U20170）及人源肺孢子虫（DQ473446）同源性均为100%（154/154、155/155）。结论 mtLSU-巢式PCR方法应用于大鼠卡氏肺孢子虫检测敏感性高,特异性强;获得与人源耶氏肺孢子虫相同的Wistar大鼠卡氏肺孢子虫mtLSU的基因序列。

mtLSU-nested polymerase chain reaction in the detection of Pheumocystis carinii in experimental rat

Objective To investigate the sensitivity and diagnostic value of nested PCR with mtLSU in the detection of Pneumocystis carinii（P.carinii） in rat and to determine the mtLSU gene sequence of P.carinii from Wiatar rat.Methods P.carinii infection model was established by subcutaneous injection of an immunodepressant dexamethasone in rats.The study was carried out in 2 groups,including a control group（1 rat） and an experimental group（10 rats）.Bronchoalveolar lavage fluid（BALF） and lung tissue from the rats were collected and assayed after week-7 by the nested PCR.The primer for mtLSU was used for the nested PCR.Microscopic examination of lung tissues and BALF smears stained with Gomori＇s methenamine silver（GMS）for P.carinii were also performed.Results Using mtLSU-nested PCR,the positive detection rate for the P.carinii DNA in the infected lung tissues and BALF was 100%（10/10）and 90%（9/10）,respectively.For the microscopic examination using GMS staining method,the positive detection rate for the P.carinii DNA in the infected lung tissues and BAL was 80%（8/10） and 60%（6/10）,respectively.The length of mtLSU was found to be 155 bp.The degree of homology of mtLSU gene from Wistar rats P.carinii was 100% as Wakefieldiae（U20170） and human（DQ473446）.Conclusion Nested-PCR is a highly sensitive and specific method for the detection of P.carinii.The mtLSU sequence from Pneumocystis jirovecii was successfully obtained using this method.