Development of a rapid detection method for Photobacterium spp. using Loop-mediated isothermal amplification (LAMP)

While the abundance of photobacteria has previously been exclusively associated with marine environments and spoilage of seafood, several recent studies have demonstrated their status as pervasive constituents of the microbiota on packaged meats. Since their ubiquitous nature has been revealed, detection of their presence on meat, their entry route into meat processing environments and prevention of their growth is a novel emerging challenge for the food industry. In this study, we have developed a highly sensitive and specific loop-mediated isothermal amplification (LAMP) assay for the detection of relevant species of photobacteria on foods, and tested its efficacy on meats. The gene encoding trimethylamine-N-oxide reductase (torA) was chosen as the target for this assay. Designed primers based on the gene sequence proved their specificity by testing 67 isolates of 5 species of photobacteria (positive) as well as 63 strains of 16 species of other common meat spoilers (negative). The optimized assay takes 2 h including sample preparation and has a detection limit of only 10-11 copies (50 fg/reaction) of the average Photobacterium (P.) genome per reaction. Its applicability could be successfully demonstrated on naturally and artificially contaminated chicken, beef and pork samples and evaluated by comparison with a culture-dependent approach using selective media and MALDI-TOF MS for identification. The developed LAMP assay revealed presence of photobacteria on one naturally contaminated chicken sample stored at 4 degrees C long before (3 days) confirmation by the culture-dependent approach. This study demonstrates that the developed LAMP assay represents a reliable and sensitive method for rapid detection of photobacteria on meats. However, its specificity would allow the applicability of the methodology to be extended to other foods, e.g. fish and seafood where presence of photobacteria is directly linked to their shelf life. The method has no requirement for specialized equipment or specially trained personal allowing an easy implementation within the quality control of the food industry. Considering the lot-to-lot variations observed on meats regarding the presence of photobacteria and the impracticality of implementing quantitative methods within the routine control, the LAMP method can simplify and reduce the workload for detection of photobacteria on high sample numbers. Consequently, producers can identify batches/plants that need more stringent control, and are provided with a tool to determine the entry route of photobacteria into the processing and distribution chain of raw meats.