Evaluation and improvement of LAMP assays for detection of Escherichia coli

serogroups O26, O45, O103, O111, O121, O145, and O157.
Deguo Wang
Henan Postdoctoral Research Base, Food and Bioengineering College, Xuchang University, Xuchang 461000, China.

Abstract: Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157 are the causative agents of human diseases, and LAMP assays have been developed for detection of the seven leading STEC serogroups.

Objective: To evaluate existing LAMP assays for detection of the seven STEC serogroups, if necessary, to improve these assays
and to promote their practical application.

Methods: Pure DNA extract from 23 strains reserved in our lab was used to evaluate the existing LAMP assays. The existing LAMP assays were modifed via adding 1% tetramethylene sulfoxide and 5% dimethyl sulfoxide as well as optimizing reaction temperature.

Results: The detection limit of the modifed LAMP assays was 0.1-1 pg per reaction, equivalent to 25-250 cfu per reaction, the non-specifc amplifcation can completely be eliminated with optimal amplifying temperature, and the modifed LAMP assays can specifcally and sensitivly amplify targeted O serogroup-specifc gene (wzx or wzy) of any concerned Escherichia coli serogroup as commercial kit Isothermal Master Mix did.

Conclusion: In conclusion, the LAMP assays were highly susceptible to non-specifc amplifcation caused by primer dimers,
and these modifed methods were free of non-specifc amplifcation and can rapidly and reliably detect the seven major Shiga
toxin-producing E. coli serogroups.

Keywords: Loop-mediated Isothermal Amplifcation (LAMP), toxin-producing Escherichia coli serogroups, non-specifc amplifcation, tetramethylene sulfoxide, dimethyl sulfoxide.