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

Author(s)

Miotto, Bruno Alonso ^[1] ; da Hora, Aline Santana ^[2] ; Taniwaki, Sueli Akemi ^[2] ; Brandao, Paulo Eduardo ^[2] ; Heinemann, Marcos Bryan ^[2] ; Hagiwara, Mitika Kuribayashi ^[1]

Affiliation

^[1] Univ Sao Paulo, Fac Med Vet & Zootecnia, Dept Clin Med, Sao Paulo, SP - Brazil

^[2] Univ Sao Paulo, Fac Med Vet & Zootecnia, Dept Med Vet Prevent & Saude Anim, Sao Paulo, SP - Brazil

• Document type: Journal article
• Source: Brazilian Journal of Microbiology; v. 49, n. 3, p. 584-590, JUL-SEP 2018.
• Web of Science Citations: 3

Abstract

Abstract A modified TaqMan real-time polymerase chain reaction targeting a 138 bp 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. (AU)

• FAPESP's process: 12/14681-7 - Evaluation of urinary shedding and identification of chronic carriers of pathogenic leptospiras in dogs holded at public or private animal shelters of São Paulo Metroplitan Area
• Grantee: Mitika Kuribayashi Hagiwara
• Support Opportunities: Regular Research Grants
• FAPESP's process: 12/13022-0 - Study of urinary shedding and identification of chronic carriers of pathogenic leptospiras in dogs kept in public or private animal shelters of Metropolitan São Paulo area
• Grantee: Bruno Alonso Miotto
• Support Opportunities: Scholarships in Brazil - Master