Rapid Identification Of Legionella Pneumophila Using Real-Time PCR Melt Curve Analysis

Objective: To develop a new method for the detection of Legionella pneumophila using melt curve based on real-time PCR.And investigate the Application value in the sputum samples and environment water samples.Methods:(1) Primers and fluorescence hybridisation probes were designed using16S rRNA gene of Legionella pneumophila.(2) The reaction system and reaction conditions were optimized and the specificity, sensitivity and repeatability" of this method were verified by detecting L. pneumophila. non-L. pneumophila and other bacteria, and doing the simulation experiment.(3) Sputum specimens of patients with pneumonia were detected using melt curve based on real-time PCR. and the result were compared with DNA sequencing method.(4) Environment water samples were culture. Biochemical phenotype appraisal. PCR. real-time PCR.DNA sequencing for identification-Results:(1) primer sequence:forward primer5’-AGGGTTGATAGGTTAAGAGC-3’. reverse primer5’-CCAACAGCTAGTTGACATCG-3’. probe sequence:anchor sequence5’-CCCATACTCGAGTCAACC (FAM)-3’.sense sequence:5’-(BHQ1) TATTATCTGACCGTCCCA G (ph)-3’.(2) Response procedures:Roche Lightcycler480realtime fluorescence PCR95℃3min;95℃10S.57℃20s,72℃30s,50cyclers:melting procedures:95℃1min:40℃2min;40℃to85℃,1℃/5s。and detect the fluorescence under the FAMc(3) The melt curve showed that the Tm of15strains L. pneumophila were57℃. the Tm of7strains non-L.pneumophila were43℃. the Tm of1strain non-L. pneumophila was45℃.19strains non-L.pneumophila and other bacteria were all negative. The sensitivity was100fg/ul. (4) Culturing the117sputum specimens, we did not get Legionella pneumophila positive samples.3sputum specimens had L. pneumophila in117patients detected with pneumonia.(5) Culturing the Environment water samples, we get22bacterial strain extracting bacterial DNA and PCR.we got20Legionella strains.Cultureing the Environment water samples, extracting bacterial DNA and PCR, we got Legionella pneumophila positive were9strains.be accroding to DNA sequencing.Extracting the Environment water samples’ DNA, and we got Legionella positive samples were4,5,8,9,13v14,15,16,17,18. Detecting by DNA sequencing,we find5,9samples were Legionella pneumophila,13,14,15samples were Legionella L. cincinnatiensis,16,17,18were Legionella longbeachae.Extracting the Environment water samples" DNA. and we did not get Legionella pneumophila positive samples.Conclusion:(1) Using the method of real-time PCR melted curve, we can detect Legionella pneumophila. and can Genotype for part Legionella.(2) In the sputum specimen.we detected3Legionella pneumophila using real-time PCR melted curve, and the result were accord with DNA sequencing method.(3)In the Environment water samples, we detect Legionella by culture. Biochemical phenotype appraisal. PCR. real-time PCR.DNA sequencing, result showed Legionella can be cultured, but for Genotype, we need by real-time PCR and DNA sequencing.(4) the method of real-time PCR melted curve was fast, sensitive, specified.and less polluting, in addition, the quenching probe fluorescence labeling was sample, the prise was low. suitable for clinical investigation and the rapid detection of L, pneumophila infection.