AUTHOR=Kreitlow Antonia , Becker André , Ahmed Marwa F. E. , Kittler Sophie , Schotte Ulrich , Plötz Madeleine , Abdulmawjood Amir 

TITLE=Combined Loop-Mediated Isothermal Amplification Assays for Rapid Detection and One-Step Differentiation of Campylobacter jejuni and Campylobacter coli in Meat Products

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2021

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.668824

DOI=10.3389/fmicb.2021.668824

ISSN=1664-302X

ABSTRACT=<p>A loop-mediated isothermal amplification (LAMP) assay system was established, allowing <italic>rplD</italic> gene-based simultaneous detection of <italic>Campylobacter jejuni</italic> and <italic>Campylobacter coli</italic> in enriched meat products. Additionally, one-step differentiation of target species on agar plates was enabled by <italic>cdtC</italic> gene- and <italic>gyrA</italic> gene-based duplex LAMP. Both the <italic>rplD</italic> and <italic>cdtC</italic>–<italic>gyrA</italic> LAMP assays amplified the target sequences in all 62 <italic>C. jejuni</italic> and 27 <italic>C. coli</italic> strains used for determining inclusivity and revealed 100% exclusivity toward 85 tested non-target species. Throughout the entire experiments, <italic>C. jejuni</italic> and <italic>C. coli</italic> strains were 100% distinguishable by melting curves of <italic>cdtC</italic> and <italic>gyrA</italic> LAMP products. After 24-h enrichment, the <italic>rplD</italic> LAMP assay reliably detected initial inoculation levels of 10–100 CFU/g in artificially contaminated minced meat. Investigation of naturally contaminated meat samples revealed a diagnostic accuracy of 95% toward real-time PCR and 94.1% toward the standard culture method applying the 24-h incubation period. Diagnostic sensitivity and specificity, and positive and negative predictive values were 89.8, 100, 100, and 91.2%, respectively, when measured against real-time PCR, and 89.6, 98.1, 97.7, and 91.2%, respectively, when measured against the standard culture method. After 48-h enrichment, the detection limit of the <italic>rplD</italic> LAMP assay improved to initial inoculation levels of 1–10 CFU/g in artificially contaminated minced meat. Applying the 48-h incubation period on naturally contaminated meat samples resulted in 100% concordant results between <italic>rplD</italic> LAMP, real-time PCR, and the standard culture method. The established LAMP assay system was proved to be suitable for rapid meat sample screening. Furthermore, it constitutes a promising tool for investigating other <italic>Campylobacter</italic> sources and could therefore make a valuable contribution to protect consumers from foodborne illness.</p>