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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。
Objective To evaluate the value of mtLSU-nested PCR in detection of Pneumocystis carinii in rats and its gene sequence. Methods The mice were infected with Pneumocystis carinii by immunosuppression with dexamethasone. In the experimental group, 10 rats in the experimental group and 1 in the control group. At the 7th week after induction, the lungs and bronchoalveolar lavage fluid (BALF ) Were used to amplify and sequence the genes shared by human and mouse Pneumocystis spores using mtLSU-nested PCR. At the same time, the lung tissues and alveolar lavage fluid samples of the experimental group were detected by microscopic examination, Evaluate the sensitivity of both methods. Results The mtLSU-nested PCR method was used to detect the pulmonary tissues and BAL in experimental infected rats. The positive rates of Pneumocystis carinii DNA were 100% (10/10) and 90% (9/10), respectively. The positive rates of GMS staining were 80% (8/10) and 60% (6/10), respectively. The mtLSU gene sequence of Pneumocystis carinii detected in Wistar rat was 155bp in length, which shared 100% identity with GenBank Pneumocystis carinii (U20170) and Pneumocystis carinii (DQ473446) in Genbank (154/154, 155/155). Conclusion The mtLSU-nested PCR method has high sensitivity and specificity for the detection of Pneumocystis carinii in rats. The mtLSU gene sequence of Pneumocystis carinii in Wistar rats was obtained.