Development of a real-time PCR for the detection of pathogenic Leptospira spp. in California sea lions

Diseases of Aquatic Organisms

Richard L. Zuerner¹  David P. Alt¹  Katherine C. Prager¹  Renee L. Galloway¹  Tracey Goldstein¹  Qingzhong Wu¹  , James O. Lloyd-Smith¹  Lori Schwacke¹ 

关键词: Sea lions;    Pathogenic Leptospira spp.;    lipL32 gene;    Real-time PCR;    Urine;    Kidney;   

DOI  :  10.3354/dao02752

学科分类：生物科学（综合）

来源: Inter-Research

【 摘 要 】

ABSTRACT: Several real-time PCR assays are currently used for detection of pathogenic Leptospira spp.; however, few methods have been described for the successful evaluation of clinical urine samples. This study reports a rapid assay for the detection of pathogenic Leptospira spp. in California sea lions Zalophus californianus using real-time PCR with primers and a probe targeting the lipL32 gene. The PCR assay had high analytic sensitivity—the limit of detection was 3 genome copies per PCR volume using L. interrogans serovar Pomona DNA and 100% analytic specificity; it detected all pathogenic leptospiral serovars tested and none of the non-pathogenic Leptospira species (L. biflexa and L. meyeri serovar Semaranga), the intermediate species L. inadai, or the non-Leptospira pathogens tested. Our assay had an amplification efficiency of 1.00. Comparisons between the real-time PCR assay and culture isolation for detection of pathogenic Leptospira spp. in urine and kidney tissue samples from California sea lions showed that samples were more often positive by real-time PCR than by culture methods. Inclusion of an internal amplification control in the real-time PCR assay showed no inhibitory effects in PCR negative samples. These studies indicated that our real-time PCR assay has high analytic sensitivity and specificity for the rapid detection of pathogenic Leptospira species in urine and kidney tissue samples.

【 授权许可】

Unknown