肺孢子虫感染大鼠模型的建立及ITS巢式PCR检测敏感性研究

目的 建立肺孢子虫感染大鼠模型并探讨ITS1-5.8S rDNA-ITS2巢式PCR检测卡氏肺孢子虫DNA的敏感性. 方法 大鼠分为实验组和对照组,实验组从第1周开始用每周2次每次每只皮下注射地塞米松1 mg的方法诱导,共8周,并分别在首次注射后第2、4、6、8周解剖大鼠制作肺印片、肺组织匀浆液及支气管肺泡灌洗液(BAL)涂片,并进行六亚甲基四胺银(Gomori's methenamine silver,GMS)染色镜检;对照组不作激素注射,并分别在0周和第10周剖杀染色检查.同时分别提取大鼠BAL和肺组织的肺孢子虫DNA进行巢式PCR扩增,比较GMS法和ITS巢式PCR检测的敏感性. 结果 实验组从第6周开始染色镜检,可见少量肺孢子虫包囊,第8周肺印片、肺组织匀浆液均检测到包囊,检出率为100%(10/10),BAL的检出率为80%(8/10),对照组则均未检出.用ITS1-5.8S rDNA-ITS2巢式PCR法均能检测出实验组第8周大鼠BAL和肺组织卡氏肺孢子虫DNA,阳性率为100%(10/10),对照组均为阴性.比较BAL标本、肺印片和肺组织匀浆液GMS染色法的检出率,BAL最低,肺组织匀浆液最高.其中20%(2/10)大鼠的BAL标本用GMS染色法未能检出,但用巢式PCR方法均能成功扩增.结论成功用地塞米松诱导法建立了肺孢子虫大鼠感染模型;ITS1-5.8S rDNA-ITS2巢式PCR检测卡氏肺孢子虫敏感性高、特异性强,可推广应用于临床诊断肺孢子虫肺炎.

Establishment of rats model of Pneumocystic carinii infection and study on the sensitivity of ITS-nested PCR assay

Objective To establish the rats model of Pneumocystis carinii infection and study on the sensitivity of ITS1-5.8S rDNA-ITS2 nested PCR. Methods SD rats were divided as experiment group and control group at random. Each experiment rat was rendered immunedepressant by subcutaneous injection of dex-amethasone from the 1 st week with 1 mg per rat twice a week for 8 weeks, and the control rats with normal feeding. P. carinii of experiment group was examined microscopically in bronchoalveolar lavage (BAL) on lung impression smears and lung tissue smears stained with Gomorismethenamine silver (GMS) at the 2nd, 4th, 6th and 8th week after injection, respectively. Those of control group was examined at the beginning of experi-ment and the 10th week. The DNAs of P. carinii were extracted beth from the BAL and lung tissues of rats. ITS1-5.8S rDNA -ITS2 of P. carinii were amplified by nested PCR. The sensitivity was compared between GMS and nested PCR. Results P. carinii could be observed from the 6th week in the experiment group and at the 8th week with 100% (10/10) positive in lung impression smears and lung tissue smears, 80% (8/10) in BAL smear. The control group was negative. Nested PCR detected the DNAs of P. carinii both from BAL and lung tissue at the 8th week with 100% positive, and all negative in control group. Comparing the detect efficiency of P. carinii among BAL, lung impression smears and lung tissue smears by GMS method, BAL was the lowest, lung tissue smears was the highest. 20% specimens of BAL couldnt be detected by GMS method, while nested PCR worked. Conclusions The rats model of P. carinii infection was successfully established by injection ofdexamethasone. ITS1-5.8S rDNA-ITS2 nested PCR is a method with high sensitivity and specificity, whichcould be potentially used to detect Pneumocystis pneumonia in clinic.