SENSOR COMPONENTS FOR PCR DETECTION OF SALMONELLA IN ALFALFA SPROUT IRRIGATION WATER

摘要

Salmonella has been associated with a number of recent outbreaks of foodborne illness involving sprouted seeds. In this research, we addressed the design of sensor components intended for use in a fully automated detection system. A real-time PCR assay was developed to detect Salmonella in sprout irrigation water. Specific detection was achieved by targeting a region of the invasion gene, invA . The detection limit for Salmonella in sterile water was approximately 400 CFU, and in sprout irrigation water the detection limit was approximately 200 CFU. Components for an automated sensing system were designed, including a thermal cycler and a fluorescent optical sensor. The thermal cycler utilized a thermoelectric module for heating and cooling the sample block and a heat sink and fan to remove heat from the module during cooling. Sample temperature was controlled to within about 1°C at each of the PCR setpoints (94°C, 55°C, and 72 ° C). The optical sensor used a laser diode (635 nm) for excitation and a bandpass interference filter (700 ±20 nm) coupled with a photodiode for fluorescence detection. The DNA dye TO-PRO-3 (642 nm excitation; 661 nm emission) was chosen to match the excitation wavelength of the laser diode. Calibration of the optical sensor with calf thymus DNA showed detection down to 0.01 µ g mL -1 , demonstrating the potential to detect down to 1 CFU if used in conjunction with PCR. When the sensor components were used to implement the real-time assay, S . Newport was detected to approximately 7.3 × 10 4 CFU in sterile water and approximately 1.5 × 10 6 CFU in sprout irrigation water. Further optimization of the assay in the sensor will be needed to improve the detection limits