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

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

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

Arquivos

2-s2.0-85024393059.pdf (1.38 MB)

Data

2017-11-02

Autores

Rubio, Marcela da Silva

Penha Filho, Rafael Antonio Casarin

Almeida, Adriana Maria de

Berchieri, Angelo

Tipo

Artigo

Direito de acesso

Acesso aberto

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°C for both biovars. The pSG amplicon (273 bp) showed a Tm of 86.2°C for S. Gallinarum and pSP amplicon (260 bp) dissociated at 84.8°C for S. Pullorum identification. The multiplex qPCR in real time showed high sensitivity and was capable of quantifying 108–101 CFU of these biovars.