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

Figure 5

Optimization of assay incubation temperature. Sample (2.5 μL) was added to 10 μL reagent mix then incubated at 60–65°C for 60 minutes, 80°C for 2 minutes, and cooled to 4°C for 10 minutes using a T100 Thermal Cycler (S/N 621BR14432). Samples used were the No Template Control (NTC) and the Positive Control (PC; 10⁶ copies/μL). Results were viewed using the EasyView™ LED Transilluminator (Manila HealthTek, Inc., Marikina, Philippines) which has a blue LED light and a 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. 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. (−/+)–Negative using the LED transilluminator but positive in 1.5% AGE.