Sensitive and rapid visual detection of Salmonella Typhimurium in milk based on recombinase polymerase amplification...

Salmonella Typhimurium (S. Typhimurium) can cause serious foodborne diseases. In this study, an assay combining recombinase polymerase amplification (RPA) with lateral flow dipsticks (LFD) was developed to detect S. Typhimurium in milk. The RPA forward primers STF1, STF2, STF3, the reverse primer STR labeled with digoxin, and the probe STProb labeled with FAM were designed and screened to produce RPA products for LFD detection. The RPA reaction volume, temperature, and time were then optimized, and the sensitivity and specificity of the developed method were analyzed. Finally, the RPA-LFD method was evaluated using milk artificially contaminated with S. Typhimurium. Results indicated that the primer pair STF1/STR is the optimal combination for detecting the bacterium. The minimum volume, shortest time, and optimal temperature of the RPA reaction were 10 μL, 10 min, and 40-42 °C, respectively. The limit of detection of RPA-LFD for detecting the genomic DNA of S. Typhimurium was 1 fg, which is 5 and 10 times lower than the corresponding limits of RPA-agarose gel electrophoresis (AGE) and PCR-AGE, respectively. Testing with 29 other foodborne bacteria as controls revealed that RPA-LFD was highly specific for S. Typhimurium. RPA-LFD can detect S. Typhimurium at concentrations as low as 1.95 CFU/mL in artificially inoculated milk samples and is thus 10 times more sensitive than PCR. Hence, the RPA-LFD assay established in this study could be a potential point-of-care/need test for S. Typhimurium, especially in areas with limited resources.