两种巢式PCR法检测实验大鼠卡氏肺孢子虫的灵敏度评价  

Evaluation of sensitivity of two nested PCR methods in detection of Pheumocystis carinii in experimental rat

作　　者：杨芳芳[1] 张小娟[1] 王鸽[2] 欧阳颐[1] 林睿[1] 黎学铭[1] 

机构地区：[1]广西壮族自治区疾病预防控制中心,南宁530021 [2]广西医科大学寄生虫教研室,南宁530021

出　　处：《应用预防医学》2012年第1期1-6,共6页Applied Preventive Medicine

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)

摘　　要：目的对ITS1-5.8S rRNA-ITS2巢式PCR法与mtLSUrRNA巢式PCR法检测大鼠卡氏肺孢子虫的敏感性进行评价。方法把大鼠随机分为7组（6组实验组,1组对照组）,每组10只,采用地塞米松免疫抑制法诱导实验组大鼠感染肺孢子虫;自第3周开始每周剖杀1组实验鼠,收集第3周至第8周实验组大鼠肺组织（LT,lung tissue）和支气管肺泡灌洗液（BALF,bronchoalveolar lavage fluid）标本。同时采用镜检法、ITS1-5.8S rRNA-ITS2巢式PCR法和mtLSUrRNA巢式PCR法对大鼠LT和BALF标本进行检测,并对结果进行统计学分析。结果采用GMS染色镜检法于第5周开始检出肺孢子虫包囊,第6、7周包囊数最多。采用ITS1-5.8S rRNA-ITS2-巢式PCR法和mtLSUrRNA巢式PCR法对实验感染大鼠LT和BALF进行检测,前者卡氏肺孢子虫阳性率分别为第3周20%（2/10）和0（0/10）;第4周70%（7/10）和10%（1/10）;第5周90%（9/10）和30%（3/10）;第6周90%（9/10）和80%（8/10）;第7周100%（10/10）和80%（8/10）;第8周40%（5/8）和40%（5/8）。后者卡氏肺孢子虫阳性率分别为第3周100%（10/10）和100（10/10）;第4周100%（10/10）和80%（8/10）;第5周100%（10/10）和50%（5/10）;第6周90%（9/10）和100%（10/10）;第7周100%（10/10）和80%（8/10）;第8周100%（8/8）和87.5%（7/8）。经？2？检验,在大鼠无明显症状阶段（3~4w）时,两种巢式PCR方法检测敏感性差异有统计学意义。有明显症状阶段（5~8w）时,其敏感性差异无统计学意义;同时统计学分析显示不同来源的标本对不同方法敏感性不同,采用mt-LSU巢式PCR检测时,LT和BALF标本敏感性差异无统计学意义,采用ITS-巢式PCR检测时,对LT检测的敏感性更高。结论 mtLSU-巢式PCR法检测实验大鼠中检出卡氏肺孢子虫敏感性高于ITS1-5.8S rRNA-ITS2-巢式PCR法。Objective To evaluate the sensitivity of ITS1-5.8S rRNA-ITS2 nested PCR and mtLSUrRNA nested PCR assays in detection of carinii（P.carinii） in experimental rat.Methods Infection of P.carinii in rats was induced by subcutaneous injection of an immunodepressant,dexamethasone.The study was carried out in 7 groups,10 rats each,including one uninfected group as a control.Samples of bronchoalveolar lavage fluid（BALF） and lung tissue（LT） from the rats were collected and assayed weekly after week 3 by the nested PCR assay using the primers for mtLSUrRNA and ITS1-5.8S rRNA-ITS2 loci of P.carinii.At the same time,microscopic examination was conducted for BALF and LT sample smears stained with Gomori＇s methenamine silver（GMS） for P.carinii.Results Cysts of P.carinii was observed under microscope at 5w post-infection,and the highest number of parasite cysts were found at 6w or 7w post-infection,.By the mtLSU nested PCR assay for LT and BALF samples,the positive rate was found to be 100% and 100%（3w）,100% and 100%（4w）,100 % and 50%（5w）,90% and 100%（6w）,100% and 80%（7w）,100% and 87.5%（8w）;respectively,and by the ITS2 nested PCR for the above two types of samples,the positive rates were 20% and 0（3w）,70% and 10%（4w）,90% and 30%（5w）,90% and 80%（6w）,100% and 80%（7w）,40% and 40%（8w）,respectively.Statistical ？？2analysis indicated that the sensitivity of mtLSU PCR was significantly higher than that of ITS2 PCR for the samples collected during the asymptomatic stage（3~4w）,while the two assays showed no significant difference in sensitivity for the samples collected at the symptomatic stage（5~8w）.Moreover,the detection sensitivity may vary due to the different collection source of the samples.When mtLSU PCR was used,the sensitivity showed no statistically significant difference for BALF and LT samples.However,the ITS2 PCR assay was more sensitive for LT samples.Conclusions The detection sensitivity of mtLSUrRNA PCR assay for P.carinii showed higher sensitivit

关 键 词：卡氏肺孢子虫 线粒体核糖体大亚基RNA基因(mtLSU) 内转录间隔区(ITS) 5.8S核糖体RNA基因(5.8SrRNA)巢式PCR 

分 类 号：R531.504[医药卫生—内科学]