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

Background: Escherichia coli is the most common pathogenic bacteria that frequently causes life-threatening opportunistic human infections, diarrhea, and septicemia in immunocompromised hosts. Methods: 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 65 degrees C for 30 minutes and 80 degrees C 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 mu M 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. Results: 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 10(0) bacterium/reaction. Conclusions: 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.