Colibacillosis is the most common disease in poultry and it is the major cause of death in flocks. Diseases caused by avian pathogenic Escherichia coli strains (APEC) result in significant financial losses to the poultry industry worldwide. Typical clinical signs related to colibacillosis are very diverse. APEC strains cause primary or secondary infections of birds. Several virulence genes were shown to be implicated in avian colibacillosis, but all determinants of pathogenicity are not explained yet, thus the control of infections and identification of the causative E. coli strains are still difficult. Nowaday we use traditional diagnostic methods for detection of APEC. These methods are time-consuming and often priceless. Due to the increasing need for rapid diagnostics, molecular methods come to the forefront, including polymerase chain reaction – PCR and loop mediated isothermal amplification – LAMP. The aim of this master thesis was to developed a method, based on LAMP, for detection of virulence-associated genes in APEC. We developed three LAMP-APEC tests for detection of three virulence-associated genes (sitA, traT and ompT). We designed six sets of primers, of which we end up choosing the most efficient one. We prepared PCR and LAMP protocols to detect the presence of target genes. The analysis revealed that both methods are comparable. We also prepared a protocol for direct detection of virulence-associated genes without prior extraction of DNA. This protocol is suitable for different types of samples (tissues of dead animal’s internal organs, feed, litter, feces and air). With LAMP we were able to detect all three virulence genes in different samples in less than 30 minutes. We also proved that LAMP is not sensitive to PCR inhibitors from animal tissue and other matrix. LAMP has proven to be specific, robust, selective, sensitive, fast and easy to use as a diagnostic tool for detection of the selected virulence genes in APEC. The big advantage is also the fact that the method can be easily performed on the sampling site.
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