A multiplex PCR assay has been developed for the identification of the six common Campylobacter taxa associated with human gastroenteritis and/or septicaemia, namely Campylobacter coli, Campylobacter fetus, Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter jejuni, Campylobacter lari and Campylobacter upsaliensis. The assay was developed using a combination of newly designed and published primers. It provided a specific PCR product for each of the five Campylobacter species and the one subspecies, and each of the PCR products was sufficiently distinguished by a difference in size by agarose gel electrophoresis. On evaluation of efficacy with 142 Campylobacter strains, the assay correctly identified all strains as 1 of the 6 Campylobacter taxa. This multiplex PCR assay is a rapid, simple and practical tool for identification of the six Campylobacter taxa commonly associated with gastroenteritis and/or septicaemia in humans, and offers an effective alternative to conventional biochemical-based assays.

No abstract

We investigated the efficacy of a loop-mediated isothermal amplification (LAMP) assay for detection of chicken meat samples naturally contaminated with Campylobacter jejuni and Campylobacter coli. A total of 144 Preston enrichment broth cultures from chicken meat samples were assessed by using the LAMP assay and conventional culture methods, which consist of a combination of Preston enrichment culturing and plating onto Butzler and modified charcoal cefoperazone deoxycholate agars. Compared with C. jejuni-C. coli isolation using the conventional culture test, the LAMP results showed 98.5% (67/68) and 97.4% (74/76) sensitivity and specificity, respectively, and the positive and negative predictive values were 97.1% (67/69) and 98.7% (74/75), respectively. The conventional culture test required more than 3 to 4 days to isolate and identify C. jejuni and C. coli in the Preston enrichment cultures. In contrast, the LAMP assay was markedly faster, requiring less than 90 min from the beginning of DNA extraction to final detection and differentiation of C. jejuni and C. coli. In total, the LAMP assay required 23.5 to 25.5 h from the beginning of the enrichment culture to final determination. These results suggest that our LAMP assay is a powerful tool for rapid, sensitive, and practical detection of C. jejuni and C. coli which may facilitate surveillance and control of C. jejuni-C. coli contamination in chicken, as well as investigations of food poisoning incidents caused by these organisms. This is the first report of a highly sensitive and specific LAMP assay to detect and differentiate C. jejuni and C. coli in chicken meat samples.

The increasing occurrence of puffer fish containing tetrodotoxin (TTX) in the Mediterranean could represent a major food safety risk for European consumers and threaten the fishing industry. The work presented herein describes the development of a new enzyme linked immunosorbent assay (mELISA) based on the immobilization of TTX through dithiol monolayers self-assembled on maleimide plates, which provides an ordered and oriented antigen immobilization and favors the antigen-antibody affinity interaction. The mELISA was found to have a limit of detection (LOD) of TTX of 0.23 mg/kg of puffer fish matrix. The mELISA and a surface plasmon resonance (SPR) immunosensor previously developed were employed to establish the cross-reactivity factors (CRFs) of 5,6,11-trideoxy-TTX, 5,11-deoxy-TTX, 11-nor-TTX-6-ol, and 5,6,11-trideoxy-4-anhydro-TTX, as well as to determine TTX equivalent contents in puffer fish samples. Results obtained by both immunochemical tools were correlated (R(2) = 0.977). The puffer fish samples were also analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the corresponding CRFs were applied to the individual TTX contents. Results provided by the immunochemical tools, when compared with those obtained by LC-MS/MS, showed a good degree of correlation (R(2) = 0.991 and 0.979 for mELISA and SPR, respectively). The mouse bioassay (MBA) slightly overestimated the CRF adjusted TTX content of samples when compared with the data obtained from the other techniques. The mELISA has been demonstrated to be fit for the purpose for screening samples in monitoring programs and in research activities.

No abstract