Rapid detection of multiple resistance genes to last-resort antibiotics in Enterobacteriaceae pathogens by recombinase polymerase amplification combined with lateral flow dipstick

Abstract

The worrying emergence of multiple resistance genes to last-resort antibiotics in food animals and human populations throughout the food chain and relevant environments has been increasingly reported worldwide. Enterobacteriaceae pathogens are considered the most common reservoirs of such antibiotic resistance genes (ARGs). Thus, a rapid, efficient and accurate detection method to simultaneously screen and monitor such ARGs in Enterobacteriaceae pathogens has become an urgent need. Our study developed a recombinase polymerase amplification (RPA) assay combined with a lateral flow dipstick (LFD) for simultaneously detecting predominant resistance genes to last-resort antibiotics of Enterobacteriaceae pathogens, including mcr-1, bla_NDM-1 and tet(X4). It is allowed to complete the entire process, including crude DNA extraction, amplification as well as reading, within 40 min at 37°C, and the detection limit is 10¹ copies/μl for mcr-1, bla_NDM-1 and tet(X4). Sensitivity analysis showed obvious association of color signals with the template concentrations of mcr-1, bla_NDM-1 and tet(X4) genes in Enterobacteriaceae pathogens using a test strip reader (R ² = 0.9881, R ² = 0.9745, and R ² = 0.9807, respectively), allowing for quantitative detection using multiplex RPA-LFD assays. Therefore, the RPA-LFD assay can suitably help to detect multiple resistance genes to last-resort antibiotics in foodborne pathogens and has potential applications in the field.

Keywords: Enterobacteriaceae; lateral flow dipstick; rapid detection; recombinase polymerase amplification; resistance genes to last-resort antibiotics.

Figure 1

RPA-LFD assay strategy. (A) DNA extraction of Enterobacteriaceae strains; (B) RPA amplification; (C) LFD lateral flow; (D) quantitative analysis.

Figure 2

Optimization of triplex RPA amplification conditions. (A) Concentration of primer/LF probe; (B) reaction temperature (1–6: 30, 35, 37, 39, 45, 50°C); (C) concentration of magnesium-acetate (1–7: 0, 2.8, 5.6, 8.4, 11.2, 14, 16.8 mM); (D) reaction time (1–10: 0, 2.5, 5, 7.5, 10, 12.5, 15, 20, 25, 30 min).

Figure 3

Specificity analysis of the triplex RPA-LFD assay. 1: Top10-pUC-mcr-1 + Top10-pUC-bla_NDM-1 + Top10-pUC-tet(X4); 2: Top10-pUC-mcr-1 + Top10-pUC-bla_NDM-1; 3: Top10-pUC-mcr-1 + Top10-pUC-tet(X4); 4: Top10-pUC-bla_NDM-1 + Top10-pUC-tet(X4); 5: Top10-pUC-mcr-1; 6: Top10-pUC-bla_NDM-1; 7: Top10-pUC-tet(X4); 8: ATCC25922; 9: negative control.

Figure 4

Sensitivity analysis of the triplex RPA-LFD assay. (A) Results of RPA-LFD assays. Blank means only the running buffer was used. (B) Standard curves. The standard linear equation and correlation coefficient (R²) between the T/C value and the logarithm of the copy number of target DNA (copies/μl).