Development of a Loop-Mediated Isothermal Amplification Assay for Rapid, Sensitive Detection of Campylobacter jejuniin Cattle Farm Samples

Campylobacter jejuniis a leading cause of bacterial foodborne disease worldwide. The detection of this organism in cattle and their environment is important for the control of C. jejunitransmission and the prevention of campylobacteriosis. Here, we describe the development of a rapid and sensitive method for the detection of C. jejuniin naturally contaminated cattle farm samples, based on real-time loop-mediated isothermal amplification (LAMP) of the hipOgene. The LAMP assay was specific (100% inclusivity and exclusivity for 84 C. jejuniand 41 non–C. jejunistrains, respectively), sensitive (detection limit of 100 fg/μl), and quantifiable (R2= 0.9133). The sensitivity of the LAMP assay was then evaluated for its application to the naturally contaminated cattle farm samples. C. jejunistrains were isolated from 51 (20.7%) of 246 cattle farm samples, and the presence of the hipOgene was tested using the LAMP assay. Amplification of the hipOgene by LAMP within 30 min (mean = 10.8 min) in all C. jejuniisolates (n =51) demonstrated its rapidity and accuracy. Next, template DNA was prepared from a total of 186 enrichment broth cultures of cattle farm samples either by boiling or using a commercial kit, and the sensitivity of detection of C. jejuniwas compared between the LAMP and PCR assays. In DNA samples prepared by boiling, the higher sensitivity of the LAMP assay (84.4%) compared with the PCR assay (35.5%) indicates that it is less susceptible to the existence of inhibitors in sample material. In DNA samples prepared using a commercial kit, both the LAMP and PCR assays showed 100% sensitivity. We anticipate that the use of this rapid, sensitive, and simple LAMP assay, which is the first of its kind for the identification and screening of C. jejuniin cattle farm samples, may play an important role in the prevention of C. jejunicontamination in the food chain, thereby reducing the risk of human campylobacteriosis.