Taqman real-time PCR assays for rapid detection of avian pathogenic Escherichia coli isolates
| Autor: | Álvaro José Celmer, Vagner Ricardo Lunge, Yara Silva Casanova, Vinicius Proença da Silveira, Fabiana de Oliveira Solla Sobral, Silvia De Carli, André Salvador Kazantzi Fonseca, Nilo Ikuta, Fernanda Kieling Moreira Lehmann |
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| Rok vydání: | 2015 |
| Předmět: |
Turkeys
Virulence medicine.disease_cause Real-Time Polymerase Chain Reaction Rapid detection Microbiology Food Animals Pathogenic Escherichia coli medicine TaqMan Escherichia coli Animals Gene Escherichia coli Infections Poultry Diseases General Immunology and Microbiology biology biology.organism_classification OmpT Virology Real-time polymerase chain reaction Animal Science and Zoology Chickens Brazil |
| Zdroj: | Avian diseases. 58(4) |
| ISSN: | 0005-2086 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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