One-Day Molecular Detection of Salmonella and Campylobacter in Chicken Meat: A Pilot Study

Abstract

Salmonella and Campylobacter ssp. are bacterial pathogens responsible for most foodborne infections in EU countries. Poultry serves as a reservoir for these pathogens, and its important role in the meat industry makes it essential to develop a rapid detection assay able to provide results in one day. Indeed, the rapid identification of foodborne pathogens is an important instrument for the monitoring and prevention of epidemic outbreaks. To date, Salmonella and Campylobacter screening is mainly conducted through molecular methods (PCR or real-time PCR) performed after 18-24 h long enrichments. In this study, we evaluated short enrichments (0, 2, 4, and 6 h) combined with a colorimetric loop-mediated isothermal AMPlification (LAMP) or real-time PCR to detect Salmonella and Campylobacter in poultry meat contaminated at different concentration levels (10¹, 10³, and 10⁵ CFU/g). Our results show that real-time PCR allows the detection of Salmonella and Campylobacter, even after shorter enrichment times than prescribed by ISO references; particularly, it detected Salmonella down to 10¹ CFU/g since T0 and Campylobacter from 10³ CFU/g since T0. Detection with LAMP was comparable to real-time PCR without the requirement of a thermal cycler and with shorter execution times. These characteristics make colorimetric LAMP a valid alternative when one-day results are needed, improving the timely identification of positive meat batches, even in the absence of specialized instrumentation.

Keywords: Campylobacter; LAMP; Salmonella; foodborne diseases; poultry.

Figure 1

(a,b) Salmonella and (c,d) Campylobacter plate count. Each sample was measured in triplicate. Mean ± SD is shown. The full dataset can be found in Table S1.

Figure 2

LAMP specificity test. The sample positivity was verified by observing the change in the color of the mix (from red to yellow). (a) LAMP for Salmonella spp. detection; (b) LAMP for Campylobacter spp. detection. Legend: 1 = S. enterica; 2 = L. monocytogenes; 3 = Y. enterocolitica; 4 = verocytotoxin-producing E. coli; 5 = C. jejuni; and 6 = negative control.

Figure 3

Ct of the first batch (a) and the second batch (b) of minced chicken meat samples contaminated with Salmonella plotted against related enrichment times (0, 2, 4, and 6 h). Ct was inversely correlated with enrichment time and bacterial concentration. No amplification was detected at the 10¹ CFU/g contamination level T0 and T2 samples in batch 2. Measurements were performed on two separate batches of minced chicken meat in triplicate. The mean ± SD of each measurement is shown. The full dataset can be found in Table S3a.

Figure 4

Detection of Salmonella DNA in contaminated minced chicken meat using colorimetric LAMP (amplification time 45′). Negative samples are red and positive samples are yellow. (a) In the first batch of minced meat, the reaction detected 10¹ CFU/g with no need for an enrichment phase. (b) For the second batch, the reaction amplified 10¹ CFU/g after 2 h of enrichment. Each sample was tested in triplicate (see Figures S3 and S5 for the complete panels).

Figure 5

Ct of the first batch (a) and the second batch (b) of minced chicken meat contaminated with Campylobacter spp. plotted against enrichment times (0, 2, 4, and 6 h). Ct was inversely correlated with the starting contamination level, but not with enrichment time, highlighting the slow-growing rate of the pathogen. No amplification was detected in 10¹ CFU/g contaminated samples. Measurements were performed on two separate batches of minced chicken meat in triplicate. The mean ± SD of each measurement is shown. The full dataset can be found in Table S3b.

Figure 6

Detection of Campylobacter spp. DNA in contaminated minced chicken meat using colorimetric LAMP (amplification time 45′). Negative samples are red and positive samples are yellow. (a) In the first batch of minced meat, the reaction detected 10¹ CFU/g after 4 h of enrichment. (b) For the second batch, 10³ CFU/g were detected with no need for enrichment. Only one out of three replicates is shown here for each sample (see Figures S7 and S9 for the complete panels).