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

Abstract

A loop-mediated isothermal amplification (LAMP) assay system was established, allowing rplD gene-based simultaneous detection of Campylobacter jejuni and Campylobacter coli in enriched meat products. Additionally, one-step differentiation of target species on agar plates was enabled by cdtC gene- and gyrA gene-based duplex LAMP. Both the rplD and cdtC–gyrA LAMP assays amplified the target sequences in all 62 C. jejuni and 27 C. coli strains used for determining inclusivity and revealed 100% exclusivity toward 85 tested non-target species. Throughout the entire experiments, C. jejuni and C. coli strains were 100% distinguishable by melting curves of cdtC and gyrA LAMP products. After 24-h enrichment, the rplD 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 rplD 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 rplD 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 Campylobacter sources and could therefore make a valuable contribution to protect consumers from foodborne illness.

A Cutoff Determination of Real-Time Loop-Mediated Isothermal Amplification (LAMP) for End-Point Detection of Campylobacter jejuni in Chicken Meat

“…Incubation time should not go beyond this point to avoid a false-positive interpretation that can occur from non-specific amplification due to the high concentration of primers commonly used in the assay. Compared with the study of cdtC – gyrA LAMP assay, the mean detection time of the assay in the detection of C. jejuni was 12 min [ 36 ].…”

“…From our review, a designed set of primers targeting the rplD gene provided 100% specificity in the detection of Campylobacter (both C. jejuni and C. coli ), and the cdtC gene- and gyrA gene-based duplex LAMP provided simultaneous detection of C. jejuni and C. coli , using melting curves for the differentiation with 100% specificity when performed using 62 C. jejuni , 27 C. coli , and 85 non-target species in the study by Kreitlow, 2021 [ 36 ]. To date, there are studies that have taken advantage of the LAMP assay’s rapidity and simplicity to develop primers that allow the simultaneous detection of multiple bacterial strains, such as the development of degenerate primers targeting the 16S rRNA sequence for the all-inclusive detection of human pathogenic Campylobacter species ( C. jejuni , C. coli , and C. lari ) or the development of duplex LAMP assays for the identification of two bacterial strains [ 36 , 40 , 50 ].…”

“…The detection limit of the LAMP assay was 100 times higher than the equivalent PCR assay. The studies of Kreitlow and Sridapan also reported the detection limit of 10 3 CFU/mL in enriched meat samples using the LAMP assays targeting the cdtC – gyrA genes ( cdtC primer set for the detection of C. jejuni and gyrA primer set for the detection of C. coli ) and 16S rRNA, respectively [ 36 , 50 ]. In another study by Yamazaki, it was reported that Preston enrichment media was used for the growth of C. jejuni contamination in chicken meat samples in combination with a three-step centrifugation procedure, reduced culture time, and increased detection sensitivity [ 51 ].…”

“…Sample preparation with an enrichment step is a limitation of this LAMP assay on rapid detection strategies that are aimed to be used for the poultry production chain, as also seen in the study by Kreitlow [ 36 ]. The total time from sample preparation to the final result was still too long compared with the kind of sample used, such as feces.…”

Immunocapture Magnetic Beads Enhanced the LAMP-CRISPR/Cas12a Method for the Sensitive, Specific, and Visual Detection of Campylobacter jejuni

“…However, PCR, qPCR, ddPCR, and other alternating temperature, amplification-based detection techniques require trained personnel, expensive equipment, and long reaction times. These requirements make them unsuitable for simple, fast, and point-of-care (POC) molecular diagnosis [14][15][16]. NGS technology has emerged as a promising approach for pathogen detection due to its high-throughput and superior sensitivity and specificity [17].…”