Establishment and application of TaqMan Real-time PCR for the detection of pathogenic Leptospira species
10.3760/cma.j.issn.0254-6450.2011.10.015
- VernacularTitle:致病性钩端螺旋体TaqMan Real-timePCR检测技术的建立及其应用
- Author:
Cui-Cai ZHANG
1
;
Xiu-Wen LI
;
Yi-Xin NIE
;
Hui-Mian YANG
;
Xiu-Gao JIANG
Author Information
1. 中国疾病预防控制中心传染病预防控制所
- Keywords:
Leptospira;
TaqMan;
Real-time polymerase chain reaction
- From:Chinese Journal of Epidemiology
2011;32(10):1018-1021
- CountryChina
- Language:Chinese
-
Abstract:
Objective To develop and evaluate a TaqMan Real-time PCR method for the detection of pathogenic Leptospira species.Methods 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,21non-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.Results 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 104 copy/μl respectively.The DNA sensitivity of Real-time PCR and conventional PCR were 100 fg/μl and 1 ng/μl respectively.The kidney tissue detection of the two methods appeared to be exactly the same.Conclusion This research project successfully developed a Real-time PCR methodology with better sensitivity and specificity for the identification of pathogenic Leptospira,using the rrs gene.
