实验感染大、小鼠和临床疑似病例肺孢子虫mt LSU rRNA基因检测

为探讨PCR技术对实验感染大、小鼠肺孢子虫肺炎(PCP)和临床疑似病例诊断的可行性。用皮下注射地塞米松法诱导SD大鼠和ICR小鼠PCP并收集临床疑似病例24h深部痰液。受试动物解剖后,制备肺印片,经瑞姬氏复合染色镜检确定肺孢子虫(Pc)感染的阳性率;同时,以针对虫体mtLSUrRNA基因设计的引物,PCR扩增鼠肺组织和支气管灌洗液(BALF),以及痰液样本中的DNA;比较实验动物样本镜检和PCR两种方法的检测结果;测定PCR的敏感性和特异性。结果表明,SD大鼠和ICR小鼠肺印片的阳性率分别为68.8%(1116)和85.7%(1821),肺组织PCR检测的阳性率分别为75%(1216)和80.9%(1721),肺泡灌洗液PCR的阳性率分别为81.2%(1316)和23.8%(521)。肺印片镜检和PCR技术检测结果无显著性差别;可见,PCR对虫体mtLSUrRNA基因的检测具有显著的特异性和敏感性,至少可以检测出0.6pg的肺孢子虫DNA;6例临床疑似病例样本中2例显示肺孢子虫PCR阳性反应。

DETECTION OF MT LSU rRNA GENE FROM PNEUMOCYSTIS IN EXPERIMENTAL INFECTED RATS,MICE AND CLINICAL SUSPICIOUS CASES

To evaluate the feasibility of PCR for diagnosis of Pneumocystis Pneumonia(PCP)in experimental infected rats and mice and clinical suspicious cases,PCP were developed in SD rats and ICR mice by quantitative subcutaneous injection of dezamethasone.Sputum from suspicious clinical PCP cases were also collected.After the infected animals were sacrificed,lung smears and bronchalveolar lavage fluid(BALF)were collected and made.Smears from the lung tissues were stained with Wright-Giemsa's compound stain and examined microscopically.Genomic DNA of the parasite in lung and BALF were also extracted and amplified by PCR,using the primers designed for the mt LSU rRNA gene.PCR results were compared with those of the microscopic examinations.The positive rates of Pneumocystis cysts in lung smears of SD rats and ICR mice were 68.8%(11/16)and 85.7%(18/21)respectively.The PCR positive rates of lung tissues from SD rats and the ICR mice were 75%(12/16)and 80.9%(17/21)separately,and that of BALF from SD rats and the ICR mice were 81.16%(13/16)and 23.8%(5/21)in turn.There is no statistical difference between the results of PCR and the lung tissues smear examination.PCR could detect 0.6 pg DNA of Pneumocystis at least which indicated the sensitivity of PCR detection. Specificity of PCR were also proved by the parallel test alone with seven parasites DNA such as Giardia lamblia.Out of 6 suspicious clinical PCP cases,2 showed positive reactions in PCR.PCR for the detection of animals infected by Pneumocystis experimentally has high specificity and sensitivity and is feasible for the diagnosis of suspicious clinical PCP cases.The results of the present experiment supply the data for the further study on the molecular detection of PCP.