Development and validation of a modified TaqMan based real-time PCR assay targeting the lipl32 gene for detection of pathogenic Leptospira in canine urine samples

A modified TaqMan real-time polymerase chain reaction targeting a 138bp fragment within the lipl32 gene was developed to identify exclusively pathogenic Leptospira spp. in dog urine samples. Thirty-five samples from dogs with suspected clinical leptospirosis and 116 samples from apparently healthy d...

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Bibliographic Details
Published in:Brazilian journal of microbiology 2018-07, Vol.49 (3), p.584-590
Main Authors: Miotto, Bruno Alonso, da Hora, Aline Santana, Taniwaki, Sueli Akemi, Brandão, Paulo Eduardo, Heinemann, Marcos Bryan, Hagiwara, Mitika Kuribayashi
Format: Article
Language:English
Subjects:
Assaying
Coefficient of variation
Confidence intervals
Deoxyribonucleic acid
Diagnostic software
Diagnostic systems
DNA
Dog
Dogs
Gene sequencing
Genomes
Leptospira
Leptospirosis
lipl32
MICROBIOLOGY
Microorganisms
Polymerase chain reaction
qPCR
Real time
Reproducibility
Ribonucleic acid
RNA
Strains (organisms)
Urine
Veterinary Microbiology
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Summary:A modified TaqMan real-time polymerase chain reaction targeting a 138bp fragment within the lipl32 gene was developed to identify exclusively pathogenic Leptospira spp. in dog urine samples. Thirty-five samples from dogs with suspected clinical leptospirosis and 116 samples from apparently healthy dogs were tested for presence of leptospiral DNA using the TaqMan-based assay. The results were compared with those from a well-established conventional PCR targeting the 16S RNA encoding gene associated with nucleotide sequencing analysis. The overall agreement between the assays was 94.8% (confidence interval [CI] 95% 88–100%). The newly developed assay presented 91.6% (CI 95% 71.5–98.5%) relative sensitivity (22[+] lipl32 PCR/24[+] 16S RNA and sequencing), 100% (CI 95% 96.3–100%) relative specificity and 98.7% accuracy (CI 95% 94.8–100%). The lipl32 assay was able to detect and quantify at least 10 genome equivalents/reaction. DNA extracted from 17 pathogenic Leptospira spp., 8 intermediate/saprophytic strains and 21 different pathogenic microorganisms were also tested using the lipl32 assay, resulting in amplification exclusively for pathogenic leptospiral strains. The results also demonstrated high intra and inter-assay reproducibility (coefficient of variation 1.50 and 1.12, respectively), thereby qualifying the newly developed assay as a highly sensitive, specific and reliable diagnostic tool for leptospiral infection in dogs using urine specimens.
ISSN:1517-8382
1678-4405
1678-4405
DOI:10.1016/j.bjm.2017.09.004