Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Hypothetical Protein Gene in Escherichia Coli Clinical Isolates

Escherichia coli is the most common pathogenic bacteria that frequently causes life-threatening opportunistic human infections, diarrhea, and septicemia in immunocompromised hosts.

This study aimed to establish a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of a hypothetical protein from an E. coli-specific gene (GenBank ID: 13702648). The gene was obtained through local and online BLAST, and specific primers were designed for this gene. Reaction conditions were optimized at 65ºC for 30 minutes and 80ºC for 2 minutes, whereas the reaction system contained 5.2 mM Mg2+, 8 U of Bst 2.0 DNA polymerase, 1.4 mM deoxyribonucleotide, and 0.2 and 1.6 µM of the outer and inner primers, respectively. The LAMP method was evaluated using 240 strains of E. coli and 150 strains of non-E. coli.

Positive reactions were observed on all 240 strains of E. coli while all non-E. coli strains were negative. Plasmids with the specific gene and mice blood with E. coli were used for sensitivity analysis. The detection limit of LAMP was 100 bacterium/reaction.

Results showed that the LAMP targeted to the hypothetical protein (GenBank ID: 13702648) is a fast, specific, sensitive, inexpensive, and suitable method for the detection of E. coli.