Rapid and simple detection of shiga toxin-producing escherichia coli (Stec) o157:H7 and stec NON-O157:H7 serotypes using colorimetric loop-mediated isothermal amplification assay

Abstract

Shiga toxin-producing Escherichia coli (STEC) detection in food is crucial for foodborne pathogen assessment, control and prevention. A colorimetric malachite green loop-mediated isothermal amplification (LAMP-MG) assay was applied to detect STEC O157:H7 and non-O157:H7. LAMP assay primers targeted E. coli O157 wzy, O157:H7 yliE and virulence genes eae, stx1 and stx2, and assay results were visualized using the naked eye. Detection limit of LAMP-MG assay was 10-100 copies/reaction, with sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 95, 99, 80, 100, and 99%, respectively compared to reference standard. STEC contamination in vegetable and fruit samples (n = 300) from fresh food markets in Bangkok, Thailand is significantly higher than that of supermarket samples (24.7 vs 11.3%; p-value = 0.0001; odds ratio = 2.18, 95% confidence interval: 1.28-3.70), with E. coli O157, O157:H7 and non-O157:H7 harboring stx2 found more often in fresh food market samples. In conclusion, LAMP-MG assay provides a simple, sensitive and rapid technique for detection of Shiga toxin-producing E. coli O157:H7 and non-O157:H7 contamination in fresh fruits and vegetables.