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

Autor(es)

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]

Afiliação do(s) autor(es)

^[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

• Tipo de documento: Artigo Científico
• Fonte: Brazilian Journal of Microbiology; v. 49, n. 3, p. 584-590, JUL-SEP 2018.
• Citações Web of Science: 3

Resumo

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)

• Processo FAPESP: 12/14681-7 - Avaliação da leptospirúria e identificação de portadores de leptospiras patogênicas em cães mantidos em abrigos públicos ou particulares da região metropolitana de São Paulo
• Beneficiário: Mitika Kuribayashi Hagiwara
• Modalidade de apoio: Auxílio à Pesquisa - Regular
• Processo FAPESP: 12/13022-0 - Avaliação da leptospirúria e identificação de portadores de leptospiras patogênicas em cães mantidos em abrigos públicos ou particulares da Região Metropolitana de São Paulo
• Beneficiário: Bruno Alonso Miotto
• Modalidade de apoio: Bolsas no Brasil - Mestrado