Development of a multiplex qPCR in real time for quantification and differential diagnosis of Salmonella Gallinarum and Salmonella Pullorum

Autor(es)

Rubio, Marcela da Silva ^[1] ; Casarin Penha Filho, Rafael Antonio ^[1] ; de Almeida, Adriana Maria ^[1] ; Berchieri Junior, Angelo ^[1]

Afiliação do(s) autor(es)

^[1] Sao Paulo State Univ FCAV UNESP, Sch Agr & Vet Sci, Sao Paulo - Brazil

• Tipo de documento: Artigo Científico
• Fonte: AVIAN PATHOLOGY; v. 46, n. 6, p. 644-651, 2017.
• Citações Web of Science: 2

Resumo

Currently there are 2659 Salmonella serovars. The host-specific biovars Salmonella Pullorum and Salmonella Gallinarum cause systemic infections in food-producing and wild birds. Fast diagnosis is crucial to control the dissemination in avian environments. The present work describes the development of a multiplex qPCR in real time using a low-cost DNA dye (SYBr Green) to identify and quantify these biovars. Primers were chosen based on genomic regions of difference (RoD) and optimized to control dimers. Primers pSGP detect both host-specific biovars but not other serovars and pSG and pSP differentiate biovars. Three amplicons showed different melting temperatures (Tm), allowing differentiation. The pSGP amplicon (97 bp) showed Tm of 78 degrees C for both biovars. The pSG amplicon (273 bp) showed a Tm of 86.2 degrees C for S. Gallinarum and pSP amplicon (260 bp) dissociated at 84.8 degrees C for S. Pullorum identification. The multiplex qPCR in real time showed high sensitivity and was capable of quantifying 10(8)-10(1) CFU of these biovars. (AU)

• Processo FAPESP: 14/05496-7 - Utilização de PCR em tempo real quantitativo para diagnóstico diferencial entre Salmonella enterica subesp. enterica sorovares enteritidis, Typhimurium e Gallinarum (biovares Gallinarum e Pullorum) em aves domésticas (Gallus Gallus)
• Beneficiário: Marcela da Silva Rubio
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 12/24017-7 - Avaliação da infecção e imunidade de linhagens de aves (Gallus gallus) suscetíveis e resistentes ao desafio por Salmonella enterica subesp. enterica sorovar Gallinarum
• Beneficiário: Rafael Antonio Casarin Penha Filho
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado