卡氏肺孢子虫特异性DNA探针的制备及其实验应用

目的制备特异、敏感的卡氏肺孢子虫(Pc)DNA探针,用于检测卡氏肺孢子虫肺炎(PCP)患者临床标本中的PcDNA。方法PCR扩增获得Pc的线粒体核糖体RNA大亚基基因,以其为模板,采用随机引物法制备半抗原地高辛标记的探针。检测探针的敏感性和特异性后,用斑点杂交法检测实验大鼠肺组织和PCP患者临床标本中的PcDNA。结果敏感性检测显示该探针可检出2pg水平的PcDNA,特异性检测显示该探针仅与PcDNA杂交,而不与伯氏疟原虫、恶性疟原虫、弓形虫、人白细胞及正常大鼠肺组织DNA反应。斑点杂交检测PCP大鼠肺组织中的PcDNA,检出率为73.7%,低于PCR结合斑点杂交的检出率94.7%。用该探针从1例肾移植术并发PCP患者的支气管肺泡灌洗液中检测到PcDNA。结论制备的PcDNA探针具有敏感性高、特异性好、无放射性污染等优点,对PcDNA有良好的检测效果。

Preparation and application of the specific DNA-probes for Pneumocystis carinii

To prepare specific and sensitive DNA probes for use in the detection of Pneumocystis carinii (Pc)DNA in the lung tissues of infected rats and the clinical specimens of patients suffered from Pc pneumonia (PCP), the large subunit mitochondrial ribosomal RNA (LSU mt rRNA) gene of Pc was amplified by PCR and used as template. Digoxigenin labeled non-radioactive probe was prepared by random priming. After the specificity and sensitivity of this probe were identified, it was used in dot hybridization to detect Pc DNA from lung tissues of the infected rats and the clinical specimens of patients with PCR. The results showed that the lowest detectable amount as determined by this probe was 2 pg. This prove only reacted with the Pc DNA, but not with DNAs from Plasmodium berghei, P.falciparium, Toxoplasma gondii, human leukocytes, and lung tissues of normal rats. The detection rate of Pc DNA from the lung tissues of rats with PCP by dot hybridization assay was 73.7%,that was significantly lower than that of PCR in combination with dot hybridization (94.7%). By using this probe,Pc DNA could be detected in the bronchoalveolar lavage fluid (BAL)of renal transplantation recipient. It is concluded that the probe prepared in the present study is proved to be sensitive, specific and non-radioactive that it can be used for the detection of Pneumocystis carinii DNA from various sources.