Taqman Real-Time PCR Assays for Rapid Detection of Avian Pathogenic Escherichia coli Isolates

• Published in: Avian diseases 2014-12, Vol.58 (4), p.628-631
• Main Authors: Ikuta, Nilo, de Oliveira Solla Sobral, Fabiana, Lehmann, Fernanda Kieling Moreira, da Silveira, Vinicius Proença, de Carli, Silvia, Casanova, Yara Silva, Celmer, Álvaro José, Fonseca, André Salvador Kazantzi, Lunge, Vagner Ricardo
• Format: Article
• Language: English
• Subjects: Animals; APEC; Aves; Brazil - epidemiology; Chickens; Escherichia coli; Escherichia coli - isolation & purification; Escherichia coli - pathogenicity; Escherichia coli Infections - microbiology; Escherichia coli Infections - veterinary; Poultry Diseases - diagnosis; Poultry Diseases - epidemiology; Poultry Diseases - microbiology; real-time PCR; Real-Time Polymerase Chain Reaction - veterinary; Research Notes; Turkeys; Virulence; virulence genes
• ISSN: 0005-2086; 1938-4351; 1938-4351
• DOI: 10.1637/10871-052414-ResNote.1

Avian pathogenic Escherichia coli (APEC) isolates are currently differentiated from nonpathogenic strains by classical PCR of virulence genes. This study improves the detection of the five main virulence genes used for APEC detection with the development of duplex and single Taqman real-time PCR to these targets. Primers and probes targeted to ompT, hlyF, iroN, iutA, and iss genes were designed and used in the implementation of single (iss) and duplex (hlyF/ompT and iroN/iutA) Taqman PCR assays. All five virulence genes of E. coli strains were successfully detected by classical and Taqman real-time (single and duplex) PCR. A panel of 111 E. coli isolates, obtained from avian samples collected in different Brazilian regions between 2010 and 2011, were further tested by both assays. Complete agreement was observed in the detection of four genes, ompT, hlyF, iron, iutA, but not for iss. This issue was addressed by combining the forward primer of the classical PCR to the new iss reverse primer and probe, resulting in complete agreement for all five genes. In total, 61 (55%) Brazilian E. coli isolates were detected as APEC, and the remaining 50 (45%) as avian fecal E. coli (AFEC). In conclusion, classical and Taqman real-time PCR presented exactly the same analytical performance for the differentiation of APEC and AFEC isolates. The developed real-time Taqman PCR assays could be used for the detection and differentiation of APEC isolates. Nota de Investigación- Métodos de PCR en tiempo real TaqMan para la detección rápida de aislamientos de Escherichia coli patógenos para las aves. Los aislamientos de Escherichia coli patógenos para las aves (con las siglas en inglés APEC) actualmente se pueden diferenciar de cepas no patógenas mediante métodos de PCR clásicos para genes de virulencia. Este studio actual mejoró la detección de los cinco genes de virulencia principales utilizados para la detección de APEC con el desarrollo métodos de PCR en tiempo real TaqMan de tipo dúplex y sencillo para estas secuencias objetivo. Los iniciadores y sondas específicas para ompT, hlyF, iroN, iutA, y los genes iss fueron diseñados y utilizados en la ejecución de un ensayo de PCR en tiempo real TaqMan simple (iss) y dúplex (hlyF/ompT and iroN/iutA). Los cinco genes de virulencia de cepas de E. coli se detectaron con éxito por PCR clásico y en tiempo real TaqMan (simple y dúplex). Un panel de 111 aislamientos de E. coli, obtenidos a partir de muestras de aves, recolectadas de difer