Rapid and visual detection of Shiga-toxigenic Escherichia coli (STEC) in carabeef meat harnessing loop-mediated isothermal amplification (LAMP)

Abstract

Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94 fg/µLand 25.8 fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12 h enrichment. The study highlights the importance of short (6-12 h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9 h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.

Keywords: Stx1; Stx2; Carabeef; Isothermal amplification; LAMP; STEC.

Fig. 1

Specificity of the stx1-LAMP assay Detection of LAMP products in tubes by addition of SYBR Green I indicating green fluorescence and their corresponding agarose gel electrophoresis indicating ladder pattern in stx-1 positive E. coli strains.Tube (1–21): NTC (No template control), DE 95 Clinical isolate, stx2 positive E. coli strain 1 A, S. aureus ATCC 43,300, E. coli O157:H7 ATCC 43,888, Proteus vulgaris P-1, Klebsiella pneumoniae ATCC 700,603, Pseudomonas aeruginosa MTCC 1688, Pasteurella multocida P52 Vaccine strain, Brucella abortus S19 Vaccine strain, Campylobacter jejuni ATCC 29,428, Edwardsiella tarda MTCC 2400, Citrobacter freundii MTCC 2956, Listeria monocytogenes ATCC 19,115, Bacillus cereus MTCC 430, Mycobacterium tuberculosis MTCC 300, Salmonella Typhimurium MTCC 3224, C. perfringens ATCC 13,124; stx1 positive E. coli 23 A, stx1 positive E. coli 558, stx1 positive E. coli C338 Agarose gel electrophoresis of stx1-LAMP amplified products Lane M: 1 kb plus DNA ladder

Fig. 2

Specificity of the stx2-LAMP assay Detection of LAMP products in tubes by addition of SYBR Green I indicating green fluorescence and their corresponding agarose gel electrophoresis indicating ladder pattern in stx-2 positive E. coli strains. Tube (1–17): NTC (No template control), stx1 positive E. coli C338, S. aureus ATCC 43,300, E. coli O157:H7 ATCC 43,888, Proteus vulgaris P-1, Klebsiella pneumoniae ATCC 700,603, Pseudomonas aeruginosa MTCC 1688, Pasteurella multocida P52 Vaccine strain, Campylobacter jejuni ATCC 29,428, Citrobacter freundii MTCC 2956, Listeria monocytogenes ATCC 19,115, Bacillus cereus MTCC 430, Salmonella Typhimurium MTCC 3224, C. perfringens ATCC 13,124; stx2 positive E. coli 1 A, stx2 positive E. coli 2B, stx2 positive E. coli C173 Agarose gel electrophoresis of stx2-LAMP amplified products; Lane M: 100 bp DNA ladder

Fig. 3

Analytical Sensitivity ofstx1-LAMP and PCR (A) Analytical sensitivity of stx1-PCR (680 bp) by agarose gel electrophoresis up to 9.4 pg DNA/µL (M: 1 kb plus DNA ladder; NTC: No Template Control) (B) Analytical sensitivity of stx1-LAMP using SYBR Green I dye and agarose gel electrophoresis showing amplification up to of 94 fg DNA/µL (M: 100 bp DNA ladder; NTC: No Template Control)

Fig. 4

Analytical Sensitivity of thestx2-LAMP and PCR (A) Analytical sensitivity of stx2-PCR (400 bp) by agarose gel electrophoresis up to 258 fg DNA/µL (B) Analytical sensitivity and visual detection of stx2-LAMP using SYBR Green I dye and Agarose gel electrophoresis of stx2-LAMP products showing amplification up to 25.8 fg DNA/µL (M: 100 bp DNA ladder; NTC: No Template Control)