Detection of Salmonella enterica subsp. enterica via Quenching of Unincorporated Amplification Signal Reporters in Loop-Mediated Isothermal Amplification

Figure 8

Assay specificity. Sample (2.5 μL) was added to 10 μL reagent mix then incubated at 65°C for 60 minutes, 80°C for 2 minutes, and cooled to 4°C for 10 minutes using T100 Thermal Cycler (S/N 621BR14432; Bio-Rad Laboratories, Inc., CA, USA). Results were viewed using the EasyViewTM LED Transilluminator (Manila HealthTek, Inc., Marikina, Philippines) which has a blue LED light and yellow plastic gel filter, and through agarose gel electrophoresis (1.5% agarose, 135 V for 25 minutes). Salmonella enterica subsp. enterica positives appear bright yellow green. Positive control panel includes the following: SPA-Salmonella enterica sv. Paratyphi A (typhoidal), SPB-Salmonella enterica sv. Paratyphi B (typhoidal), STY-Salmonella enterica sv. Typhi (typhoidal), and SNT-Salmonella enterica sv. Typhimurium (nontyphoidal). Negative control panel includes the following: Pa–Pseudomonas aeruginosa, Lm–Listeria monocytogenes, Kp–Klebsiella pneumoniae, Ec–Escherichia coli, Ef–Enterococcus faecalis, Se–Staphylococcus epidermidis (Gram-positive bacteria), and Sa–Staphylococcus aureus. Samples were assayed in triplicate. All images were taken using a Canon EOS 750D camera. Interpretation of results are as follows: (+)–Positive using the LED transilluminator and positive in 1.5% AGE; (−)–Negative using the LED transilluminator and negative in 1.5% AGE.