Loop-mediated isothermal amplification: rapid molecular detection of virulence genes associated with avian pathogenic Escherichia coli in poultry

摘要

Infections with pathogenic Escherichia coli can lead to different animal- and human-associated diseases. E. coli infections are common in intensive poultry farming, and important economic losses can be expected during infections with avian pathogenic E. coli (APEC) strains followed by colibacillosis. Loop-mediated isothermal amplification (LAMP) assays were developed for rapid detection of 3 APEC-associated virulence genes: sitA, traT, and ompT. All 3 LAMP assays are shown to be specific, repeatable, and reproducible. High sensitivities of the assays are shown, where as few as 1,000 bacterial cells/mL can be detected in different matrices. On-site applicability of this LAMP method is demonstrated through testing of different sample types, from animal swabs and tissues, and from environmental samples collected from 6 commercial poultry farms. All 3 virulence genes were detected at high rates (above 85%) in samples from layer and broiler chickens with clinical signs and, interestingly, high prevalence of those genes was detected also in samples collected from clinically healthy broiler flock (above 75%) while lower prevalence was observed in remaining 3 clinically healthy chicken flocks (less than 75%). Importantly, these virulence genes were detected in almost all of the air samples from 11 randomly selected poultry houses, indicating air as an important route of E. coli spread. Three LAMP assays that target APEC-associated virulence genes are shown to be sensitive and robust and are therefore applicable for rapid on-site testing of various sample types, from animal swabs to air. This on-site LAMP testing protocol offers rapid diagnostics, with results obtained in 35min, and it can be applied to other important microorganisms to allow the required prompt measures to be taken.