Sensitive and Specific Loop-Mediated Isothermal Amplification Assays for Detection of Salmonella, CTX-M-1 Group Genes, mph(A), and ermB in Stool and Blood Samples Based on Orange to Green Visible Dye.

Abstract

Salmonella is a globally prevalent foodborne bacterium, and ceftriaxone and azithromycin have been regarded as drugs of choice for treating Salmonella infections, particularly in children. With the growing incidence of ceftriaxone and azithromycin resistance in Salmonella, there is an urgent requirement for a rapid and dependable gene testing approach to enhance the efficacy of treating Salmonella infections. Utilizing the orange to green visible dye approach, this study developed loop-mediated isothermal amplification (LAMP) assays for the sensitive and specific detection of Salmonella, ceftriaxone and azithromycin resistance genes (including CTX-M-1 group, mph(A), and ermB genes) in stool and blood samples. The specificity and sensitivity of primers during the LAMP assays for detection of Salmonella, CTX-M-1 group, mph(A), and ermB genes were determined in this study. The detection threshold for Salmonella was found to be 1.5 × 103 colony-forming units (CFU)/mL, while it was 1.5 × 102 CFU/mL for CTX-M-1 group genes (including blaCTX-M-3, blaCTX-M-15, and blaCTX-M-55), 1.5 × 102 CFU/mL for mph(A), and 1.5 × 102 CFU/mL for ermB, showing 10-103-fold, 103-fold, and 105-fold increased sensitivity compared with the polymerase chain reaction assay, respectively. Results indicated that the LAMP primers designed for Salmonella, CTX-M-1 group, mph(A), and ermB genes possess high specificity (100%) and sensitivity (over 94%). This novel approach advocates its application in detecting Salmonella, CTX-M-1 group, mph(A), and ermB genes.