Fig 1.
Schematic depiction of d-LAMP-LFB for simultaneous detection of Campylobacter and Salmonella spp.
Table 1.
Nucleotide sequences of d-LAMP-LFB primers and probes used in this study.
Fig 2.
Detection and differentiation of LAMP products visualized by 2% agarose gel electrophoresis in comparison to LFB.
(A) Electrophoretic analysis represented the distinctive ladder-like patterns of LAMP amplification products for S: S. Typhimurium; C: C. jejuni; SC: S. Typhimurium + C. jejuni; M: 2-log DNA ladder 100 bp; Blank: the reaction with 2 μl sterile distilled water. (B) LFB analysis showed the simultaneous detection of multiple targets with distinguishing by visualizing band that appear on the C: control line; T1: test line 1 of C. jejuni; T2: test line 2 of S. Typhimurium.
Fig 3.
The specificity of d-LAMP-LFB assay for detection of different strains of 20 ng each of DNA templates.
1: S. Agona DMST 10638; 2: S. Abony DMST 21863; 3: S. Anatum DMST 16870; 4: S. Arizonae DMST 22439; 5: S. Bangkok DMST 7121; 6: S. Bergen DMST 10895; 7: S. Cerro DMST 17381; 8: S. Derby DMST 8535; 9: S. Enteritidis DMST 15676; 10: S. Gallinarum DMST 15968; 11: S. Hvittingfoss DMST 15681; 12: S. Mbandaka DMST 17377; 13: S. Newport DMST 15675; 14: S. Panama DMST 10640; 15: S. Paratyphi B DMST 28118; 16: S. Poona DMST 15679; 17: S. Schwarzengrund DMST 17364; 18: S. Senftenberg DMST 17013; 19: S. Stanley DMST 16874; 20: S. Typhi DMST 5784; 21: S. Typhimurium ATCC 23566; 22: S. Typhimurium DMST 562; 23: S. Wandsworth DMST 19204; 24: S. Waycross DMST 19205; 25: C. jejuni DMST 15190; 26: C. coli DMST 18034; 27: C. lari DMST 17953; 28: B. cereus ATCC 14579; 29: E. aerogenes DMST 2720; 30: E. coli DMST 703; 31: E. coli DMST 4212; 32: L. monocytogenes DMST 17303; 33: S. boydii DMST 30245; 34: S. aureus ATCC 25923; 35: S. epidermidis DMST 15505; 36: S. haemolyticus DMST 15511; 37: V. cholera DMST 2873; 38: V. vulnificus DMST 21245; 39: Y. enterocolitica DMST 8012; 40: blank control.
Fig 4.
Analytical sensitivity of s-LAMP-LFB assay for a single target detection of (A) C. jejuni or (B) S. Typhimurium using each of serial dilution of target DNA. (C) Sensitivity limit of d-LAMP-LFB assay applied for simultaneous detection of both pathogens using 10-fold serial dilution of mixed genomic DNA ranged from 100 ng -1 fg per reaction. C: control line; T1: test line 1 of C. jejuni; T2: test line 2 of S. Typhimurium.
Fig 5.
Lowest inoculated detection limit of s-LAMP-LFB assay for a single target detection of (A) C. jejuni or (B) S. Typhimurium in artificially contaminated raw chicken meat samples after 24 h enrichment. (C) Lowest inoculated detection limits of d-LAMP-LFB assay for simultaneous detection of both pathogens. Positive: positive control; No spike: non-spiked bacterial; Blank: blank control; C: control line; T1: test line 1 of C. jejuni; T2: test line 2 of S. Typhimurium.
Fig 6.
Evaluation of d-LAMP-LFB by detecting of Salmonella spp. and Campylobacter spp. in naturally contaminated raw chicken meat samples.
LFB: 1–30 obtained from retail markets; LFB 26 showed false-negative result for Campylobacter spp.; LFB 29 and 30 showed false-positive result for Salmonella spp.; P: positive control (C. jejuni DMST 15190 + S. Typhimurium ATCC 23566); N: blank control; * Indicated weakly positive signals; “+”: the culture test result is positive; “-”: the culture test result is negative.