Development and evaluation of loop-mediated isothermal amplification assay for the rapid detection of Escherichia coli and its microbial toxin

Abstract

Objective To establish and optimize a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Escherichia coli and its microbial toxin. Methods The LAMP reaction system and reaction conditions were determined by optimizing LAMP reaction, and the optimized LAMP system was used for the detection. Results Primers targeting shiga toxin (stx) gene and O157 antigen gene rfbe were designed. The established and optimized LAMP amplification system contained 1.2 mmol/L dNTPs, 10 mmol/L MgSO4, 0.4 mol/L betaine, 1 μl 10×Bst DNA polymerase Buffer, 8 U Bst DNA polymerase fragment, 2 μl DNA template, and the ratio of inner-primer (FIP and BIP) and outer-primer (F3 and B3) were 8∶1. Time and temperature for LAMP was 60 min, 60 ℃. The sensitivity was 103 times higher than polymerase chain reaction (PCR), reached 5×101 CFU/ml. When LAMP was applied to 19 reference strains, 102 EHEC strains, the specification was 100% while identification rate of rfbe, stx1 and stx2 gene reached 100%, 95.2%, 92.9%. Conclusions The LAMP method showed a promising prospect for the rapid detection of common nosocomial pathogens microbial toxin. Key words: Enterohemorrhagic escherichia coli/MI; Nucleic acid amplification techniques; Shiga toxin/GE; Bacterial proteins/GE