Comparison of Real-Time PCR Assays for Detection of Pathogenic Leptospira spp. in Blood and Identification of Variations in Target Sequences

被引:108
作者
Bourhy, Pascale [1 ,2 ]
Bremont, Sylvie [1 ,2 ]
Zinini, Farida [1 ,2 ]
Giry, Claude [3 ]
Picardeau, Mathieu [1 ,2 ]
机构
[1] Inst Pasteur, Unite Biol Spirochetes, F-75724 Paris 15, France
[2] Inst Pasteur, Ctr Natl Reference Leptospiroses, F-75724 Paris 15, France
[3] Ctr Hosp Mayotte, Mayotte, France
关键词
DIFFERENTIATION; GROWTH; IMPACT;
D O I
10.1128/JCM.02452-10
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
Leptospirosis is considered an underdiagnosed disease. Although several PCR-based methods are currently in use, there is little information on their comparability. In this study, four quantitative real-time PCR (qPCR) assays (SYBR green and TaqMan chemistries) targeting the secY, lfb1, and lipL32 genes were evaluated as diagnostic assays. In our hands, these assays can detect between 10(2) and 10(3) bacteria/ml of pure culture, whole-blood, plasma, and serum samples. In three independent experiments, we found a slightly higher sensitivity of the PCR assays in plasma than in whole blood and serum. We also evaluated the specificity of the PCR assays on reference Leptospira strains, including newly described Leptospira species, and clinical isolates. No amplification was detected for DNA obtained from saprophytic or intermediate Leptospira species. However, among the pathogens, we identified sequence polymorphisms in target genes that result in primer and probe mismatches and affect qPCR assay performance. In conclusion, most of these assays are sensitive and specific tools for routine diagnosis of leptospirosis. However, it is important to continually evaluate and, if necessary, modify the primers and/or probes used to ensure effective detection of the circulating Leptospira isolates.
引用
收藏
页码:2154 / 2160
页数:7
相关论文
共 24 条
[1]   Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials [J].
Ahmed, Ahmed ;
Engelberts, Mirjam F. M. ;
Boer, Kimberly R. ;
Ahmed, Niyaz ;
Hartskeerl, Rudy A. .
PLOS ONE, 2009, 4 (09)
[2]   Multilocus sequence typing method for identification and genotypic classification of pathogenic Leptospira species [J].
Ahmed N. ;
Manjulata Devi S. ;
de los Á Valverde M. ;
Vijayachari P. ;
Machang'u R.S. ;
Ellis W.A. ;
Hartskeerl R.A. .
Annals of Clinical Microbiology and Antimicrobials, 5 (1)
[3]   Isolation and Characterization of New Leptospira Genotypes from Patients in Mayotte (Indian Ocean) [J].
Bourhy, Pascale ;
Collet, Louis ;
Clement, Sabine ;
Huerre, Michel ;
Ave, Patrick ;
Giry, Claude ;
Pettinelli, Francois ;
Picardeau, Mathieu .
PLOS NEGLECTED TROPICAL DISEASES, 2010, 4 (06)
[4]   A century of Leptospira strain typing [J].
Cerqueira, Gustavo M. ;
Picardeau, Mathieu .
INFECTION GENETICS AND EVOLUTION, 2009, 9 (05) :760-768
[5]  
Dufour B, 2008, REV SCI TECH OIE, V27, P529, DOI 10.20506/rst.27.2.1817
[6]  
ELLINGHAUSEN HC, 1965, AM J VET RES, V26, P45
[7]   Asymptomatic Renal Colonization of Humans in the Peruvian Amazon by Leptospira [J].
Ganoza, Christian A. ;
Matthias, Michael A. ;
Saito, Mayuko ;
Cespedes, Manuel ;
Gotuzzo, Eduardo ;
Vinetz, Joseph M. .
PLOS NEGLECTED TROPICAL DISEASES, 2010, 4 (02)
[8]   DIFFERENTIATION OF PATHOGENIC AND SAPROPHYTIC LEPTOSPIRES .I. GROWTH AT LOW TEMPERATURES [J].
JOHNSON, RC ;
HARRIS, VG .
JOURNAL OF BACTERIOLOGY, 1967, 94 (01) :27-&
[9]  
KITTEN T, 1992, GENETICS, V132, P311
[10]   Urban epidemic of severe leptospirosis in Brazil [J].
Ko, AI ;
Reis, MG ;
Dourado, CMR ;
Johnson, WD ;
Riley, LW .
LANCET, 1999, 354 (9181) :820-825