Abstract：To analyse the transcription level alteration of lvgA gene in Legionella pneumophila after infecting host cells and establish a real-time fluorescent quantitative RT-PCR to detect L.pneumophila based on virulence gene(lvgA).The structure domains,transmembrane regions have been analyzed through using on-line database of NCBI,Pfam and TMHMM Server v.2.0.The primers have been worked out according to the DNA sequence of lvgA by ourselves.The lvgA gene was amplified from the total cell DNA of L.pneumophila by PCR,and then inserted it into the coloning vector pMD18-T.The sequence of inserted fragment was sequenced,and the lvgA sequences from different L.pneumophila species have been compared through cltust W software.The change of lvgA gene transcription levels of L.pneumophila after incubation with THP-1.1 cells was detecred by real-time fluorescence quantitative RT-PCR.Real-time quantitative RT-PCR assay based on lvgA gene for quick detection of L.pneumophila in peripheral blood of people who was infected by L.pneumophila.The prediction result suggested that lvgA located at the outside of outermembrane.The similarities of nucleotide and amino acid sequences of lvgA genes from different L.pneumophila species were 96%~99% and 98%~99%,respectively.The transcription level of lvgA gene was remarkably up-regulated after infecting the host cells.The detection result of real-time fluorescent quantitative RT-PCR were positive for all the specimens of peripheral blood collected from the L.pneumophila infected people.The lvgA gene conserved presented in different species of L.pneumophila.The alteration of lvgA gene transcription level suggests that it is an important virulence factor for L.pneumophila.The real-time fluorescent quantitative RT-PCR has been established for detecting L.pneumophila,and the advantages including quickness,stability,sensitivity and specificity,which indicats that this method can be used for clinical laboratory diagnosis of L.pneumophila infection.
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