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目的利用针对嗜肺军团菌种特异性16SrRNA和军团菌致病基因—巨噬感染增强因子(mip)序列探针原位分子杂交结合酪酰胺放大技术(Tyramide signal amplification method,TSA)建立军团菌性肺炎组织中嗜肺军团菌的快速、特异性检验方法。方法设计特异性针对嗜肺军团菌种特异性16SrRNA、mip序列探针,分别采用荧光、地高辛标记探针对嗜肺军团菌性豚鼠肺炎模型支气管肺泡灌洗液、肺组织进行原位杂交结合TSA检验嗜肺军团菌。结果荧光原位杂交显示支气管肺泡灌洗液细胞中存在16SrRNA、mip序列双杂交阳性杆状细菌;地高辛标记原位杂交结合TSA显示肺组织中也存在杂交阳性杆状细菌,分布于肺泡壁与细胞内,整个检验周期小于24h。结论结果表明采用荧光、地高辛标记探针结合TSA的原位杂交能迅速准确灵敏检验出肺组织、支气管肺泡灌洗液中的嗜肺军团菌,是一种快速、特异性检验方法。
OBJECTIVE To establish Legionella pneumophila by using in situ hybridization and Tyramide signal amplification method (TSA) against Legionella pneumophila species-specific 16S rRNA and Legionella pathogenicity gene-macrophage infection enhancer (mip) sequence probe Rapid and specific detection of Legionella pneumophila in pneumonia. Methods The specific 16S rRNA and mip sequence probes specific to Legionella pneumophila were designed and used for the in situ hybridization of bronchoalveolar lavage fluid and lung tissue of Legionella pneumophila pneumonia model by fluorescence and digoxin labeling probe respectively Legionella pneumophila combined with TSA test. Results Fluorescence in situ hybridization showed 16SrRNA and mip sequence-positive two-hybrid baculovirus positive cells in bronchoalveolar lavage fluid. In situ hybridization with digoxigenin and TSA showed that there were also positive hybrid bacilli in lung tissue, which were distributed in the alveolar wall Within the cell, the entire test cycle is less than 24h. Conclusion The results showed that the rapid and accurate detection of Legionella pneumophila in lung tissue and bronchoalveolar lavage fluid by in situ hybridization with fluorescent and digoxigenin-labeled probe combined with TSA is a rapid and specific test method.