Rapid molecular detection of salmonella from produce using real-time PCR

Author

Nathan D. Miller, University of Tennessee

Date of Award

5-2009

Degree Type

Thesis

Degree Name

Master of Science

Major

Food Science and Technology

Major Professor

Doris D'Souza

Abstract

Recent outbreaks of Salmonella linked to fresh produce emphasize the need for rapid and sensitive assays to help control outbreaks. Reverse-transcriptase PCR (RT-PCR) detects the presence of mRNA (shorter half-life than DNA), with greater potential of detecting viable cells. Rapid real-time methods using fluorescent dyes and probes simultaneously detect and confirm the presence of target nucleic acid, eliminating the need for gel electrophoresis. The objective of this research was to rapidly detect Salmonella Typhimurium from spiked lettuce, tomatoes, and peppers using real-time RT-PCR. Washed and ultraviolet light treated lettuce (~25gram), tomato (~100g), and peppers (~130g) samples were inoculated with high (10⁸ to 10⁶ CFU) and low (10³ to 10¹CFU) Salmonella Typhimurium overnight cultures. Samples were then rinsed or hand massaged with 225 ml 0.05 M glycine-saline buffer containing 0.05% Tween and 3% beef extract.Un-inoculated washed produce and sterile buffer were used as negative controls; with S. Typhimurium as a positive control. RNA was extracted from each sample using the Qiagen RNeasy Mini Kit. RT-PCR was carried out using a SYBR Green I RT-PCR kit with previously described Salmonella specific invA gene primers and an internal amplification control (IAC) to eliminate false negatives. Reaction conditions were RT at 50 degree Celsius/40min; PCR at 95 degree Celsius /45s, 58 degree Celsius /45s, 72 degree Celsius /45s for 40 cycles followed by melt temperature(Tm) analysis in a BioRad iCycler to determine specific invA product (~Tm=87.5 degree Celsius) and IAC (Tm=82 degree Celsius). To improve detection sensitivity of low inocula, spiked lettuce, tomatoes, and peppers were pre-enriched in buffered peptone water for 6 hours at 37 degree Celsius, followed by RNA extraction and RT-PCR detection. Each experiment was repeated twice.Real-time RT-PCR after 6-h pre-enrichment, Qiagen RNA extraction and the SYBR Green I kit gave Salmonella detection up to 10³ CFU/25g from lettuce, 10⁴ CFU/~130g from tomatoes, and 10⁴ CFU/25g from peppers. Without enrichment, detection limits were 10⁶ CFU/25g for lettuce, 10⁷ CFU/25g for tomatoes, and 10⁷ CFU/25g for peppers. Sensitive and rapid detection of Salmonella from spiked lettuce, tomatoes, and peppers could still be obtained within one day (~2 working shifts).

Recommended Citation

Miller, Nathan D., "Rapid molecular detection of salmonella from produce using real-time PCR. " Master's Thesis, University of Tennessee, 2009.
https://trace.tennessee.edu/utk_gradthes/5714