Purpose: The purpose of this study was to assess the limit of detection and inclusivity/exclusivity in the development of a new rapid diagnostic assay for the detection of Campylobacter spp. in food matrices.
Methods: A rapid isothermal nucleic acid amplification assay targeting a specific, conserved 16s rRNA sequence within Campylobacter spp. has been developed. A simple lysis reaction releases target rRNA. Fifty µl of the lysate is added to the lyophilized reagent. A reverse transcriptase converts rRNA to DNA with amplification occurring at a constant 56°C. After amplification, a molecular beacon probe is used to detect amplified product.
Results: The ANSR™ Campylobacter spp. assay was shown to be inclusive for C. jejuni, C. coli, and C. lari, and exclusive against other genera tested. In an enrichment study, 30 ml of poultry rinse BPW spiked with C. jejuni (n=4), ranging from 0.73-1.56 CFU/ml, were all able to be detected after 16 hour enrichment in 2X Bolton’s broth incubated at 42°C. Naturally occurring Campylobacter in poultry rinses was detected in samples that were tested prior to incubation. Upon plating onto CCA, it was found that the numbers of Campylobacter in these samples ranged from 10-55 CFU/ml. Turkey swabs were tested according to the USDA-MLG. Three were negative on CCA and ANSR after 48 hours incubation, one was positive after 24 hours while another was positive after 36 hours, reaching titers of 4.3x10e3 CFU/ml and 1.3x103CFU/mL, respectively.
Significance: The new isothermal amplification assay provides the user with a simple, fast and effective tool for the detection of Campylobacter spp. in food matrices.