Validation of absolute quantitative real-time PCR for the diagnosis of Streptococcus agalactiae in fish

Autor(es)

Sebastiao, Fernanda de A. ^[1] ; Lemos, Eliana G. M. ^[2] ; Pilarski, Fabiana ^[1]

Afiliação do(s) autor(es)

^[1] Univ Estadual Paulista, UNESP, Ctr Aquicultura, BR-14884900 Jaboticabal, SP - Brazil

^[2] UNESP Univ Estadual Paulista, Dept Technol, Fac Ciencias Agr & Vet, BR-14884900 Jaboticabal, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: Journal of Microbiological Methods; v. 119, p. 168-175, DEC 2015.
• Citações Web of Science: 10

Resumo

Streptococcus agalactiae (GBS) are Gram-positive cocci responsible for substantial losses in tilapia fish farms in Brazil and worldwide. It causes septicemia, meningoencephalitis and mortality of whole shoals that can occur within 72 h. Thus, diagnostic methods are needed that are rapid, specific and sensitive. In this study, a pair of specific primers for GBS was generated based on the cfb gene sequence and initially evaluated by conventional PCR. The protocols for absolute quantitative real-time PCR (qPCR) were then adapted to validate the technique for the identification and quantification of CBS isolated by real-time detection of amplicons using fluorescence measurements. Finally, an infectivity test was conducted in tilapia infected with CBS strains.Total DNA from the host brain was subjected to the same technique, and the strains were re-isolated to validate Koch's postulates. The assay showed 100% specificity for the other bacterial species evaluated and a sensitivity of 367 gene copies per 20 mg of brain tissue within 4 h, making this test a valuable tool for health monitoring programs. (C) 2015 Elsevier B.V. All rights reserved. (AU)

• Processo FAPESP: 11/07951-5 - Validação de técnicas moleculares para o diagnóstico de bactérias em peixes, visando redução de tempo e custo
• Beneficiário: Fernanda de Alexandre Sebastião
• Modalidade de apoio: Bolsas no Brasil - Doutorado