Establishment and application of TaqMan Real-time PCR for the detection of pathogenic Leptospira species

• Published in: Zhōnghuá liúxíngbìng zázhì Vol. 32; no. 10; p. 1018
• Main Authors: Zhang, Cui-cai, Li, Xiu-wen, Nie, Yi-xin, Yang, Hui-mian, Jiang, Xiu-gao
• Format: Journal Article
• Language: Chinese
• Published: China 01.10.2011
• Subjects: Humans; Leptospira - isolation & purification; Leptospirosis - diagnosis; Real-Time Polymerase Chain Reaction - methods; Sensitivity and Specificity
• ISSN: 0254-6450

To develop and evaluate a TaqMan Real-time PCR method for the detection of pathogenic Leptospira species. rrs gene of part fragment on 16S rRNA was used to design primers and TaqMan probe. The target gene was cloned into vector pMD19-T in order to make the standard curve and be used for quality control. To determine the specificity and specificity, DNA from Chinese Leptospira strains belonging to 15 pathogenic reference strains, 21 non-pathogenic reference strains, and 50 different serotypes of pathogenic isolates as well as 27 other micro-organisms were included in this study. Eight serial DNA dilutions from pathogenic Leptospira and DNA from 25 kidney tissues were detected by Real-time PCR and conventional PCR simultaneously. A Real-time PCR methodology was developed and optimised. All the pathogenic Leptospira gave a positive amplification. Non-pathogenic Leptospira and all the other micro-organisms were not amplified. The plasmid sensitivity of Real-time PCR and conventional PCR were 10 copy/µl and 10(4)c