counting mycobacteria in infected human cells and mouse tissue a comparison between qpcr and cfu计数分枝杆菌感染人类细胞和小鼠组织中比较qpcr和cfu.pdfVIP

Counting Mycobacteria in Infected Human Cells and Mouse Tissue: A Comparison between qPCR and CFU 1,2 3 1 3 ˚ 1,2 Sharad Pathak *, Jane A. Awuh , Nils Anders Leversen , Trude H. Flo , Birgitta Asjø 1 Section for Microbiology and Immunology, The Gade Institute, University of Bergen, Bergen, Norway, 2 Department of Microbiology and Immunology, Haukeland University Hospital, Bergen, Norway, 3 Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway Abstract Due to the slow growth rate and pathogenicity of mycobacteria, enumeration by traditional reference methods like colony counting is notoriously time-consuming, inconvenient and biohazardous. Thus, novel methods that rapidly and reliably quantify mycobacteria are warranted in experimental models to facilitate basic research, development of vaccines and anti- mycobacterial drugs. In this study we have developed quantitative polymerase chain reaction (qPCR) assays for simultaneous quantification of mycobacterial and host DNA in infected human macrophage cultures and in mouse tissues. The qPCR method cannot discriminate live from dead bacteria and found a 10- to 100-fold excess of mycobacterial genomes, relative to colony formation. However, good linear correlations were observed between viable colony counts and qPCR results from infected macrophage cultures (Pearson correlation coefficient [r] for M. tuberculosis = 0.82; M. a. avium = 0.95; M. a. paratuberculosis = 0.91). Regression models that predict colony counts from qPCR data in infected macrophages were validated empirically and showed a hig