Development of a LAMP Assay Targeting the rfbE Gene for Rapid Detection of Escherichia coli O157:H7

Abstract

Infections with Escherichia coli (E. coli) O157:H7 can lead to severe health complications. This pathogen is commonly found in contaminated meat and fresh produce, posing significant public health risks. The Loop-Mediated Isothermal Amplification (LAMP) method offers a rapid and accessible alternative to conventional nucleic acid amplification techniques, making it particularly suitable for on-site diagnostic systems. This study aimed to design a quick method using LAMP to detect the rfbE gene of E. coli O157:H7. E. coli ATCC 43888 was used as the positive control, while Candida albicans ATCC 10231, E. coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Staphylococcus aureus ATCC 29213, and Pseudomonas aeruginosa ATCC 27853 served as negative controls. Positive and negative controls were tested 10 times each for both analyses. Following DNA isolation, real-time PCR and LAMP were performed and compared with culture methods. For the positive control strain (E. coli ATCC 43888) at 10 CFU/mL, positivity was detected in 8 out of 10 samples by real-time PCR and in 7 out of 10 samples by LAMP. The sensitivity, specificity, negative predictive value, and positive predictive value of LAMP and real-time PCR were 95.00%–96.67%, 100%–100%, 94.34%–96.15%, and 100%–100%, respectively. In conclusion, our study successfully developed a rfbE gene-specific LAMP kit for E. coli O157, demonstrating comparable sensitivity and specificity to real-time PCR and culture methods. This kit can be effectively utilized in resource-limited settings.