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Evaluation and improvement of LAMP assays for detection of Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157

Deguo Wang

Abstract


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 modified via adding 1% tetramethylene sulfoxide and 5% dimethyl sulfoxide as well as optimizing reaction temperature.
Results: The detection limit of the modified LAMP assays was 0.1-1 pg per reaction, equivalent to 25-250 cfu per reaction, the non-specific amplification can completely be eliminated with optimal amplifying temperature, and the modified LAMP assays can specifically and sensitivly amplify targeted O serogroup-specific 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-specific amplification caused by primer dimers, and these modified methods were free of non-specific amplification and can rapidly and reliably detect the seven major Shiga toxin-producing E. coli serogroups.

Keywords: Loop-mediated Isothermal Amplification (LAMP), toxin-producing Escherichia coli serogroups, non-specific amplification, tetramethylene sulfoxide, dimethyl sulfoxide.




AJOL African Journals Online