development and validation of a real-time pcr for detection of pathogenic leptospira species in clinical materials开发和验证一个实时pcr检测致病性钩端螺旋体物种在临床资料.pdfVIP

Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials 1 1 1 2 1 Ahmed Ahmed , Mirjam F. M. Engelberts , Kimberly R. Boer , Niyaz Ahmed , Rudy A. Hartskeerl * 1 WHO/FAO/OIE and National Collaborating Centre for Reference and Research on Leptospirosis and Section of Epidemiology, Department of Biomedical Research, Royal Tropical Institute (KIT), Amsterdam, The Netherlands, 2 Pathogen Biology Laboratory, School of Life Sciences, University of Hyderabad, Hyderabad, India Abstract Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specific