sybr-green-i and Salmonella-Food-Poisoning

Outbreaks of Salmonella linked to fresh produce emphasize the need for rapid detection methods to curb the spread of foodborne pathogens. Reverse-transcriptase-polymerase chain reaction (RT-PCR) detects the presence of mRNA (shorter half-life than DNA), with greater potential of detecting viable pathogens. Real-time RT-PCR eliminates the need for gel electrophoresis and significantly enhances the speed of detection (<1 day) compared with traditional methods (>5 days). The objectives of this research were to apply real-time SYBR Green I-based RT-PCR to detect Salmonella from jalapeño and serrano peppers spiked with low and high inocula of Salmonella. Inoculated and uninoculated peppers were rinsed with water and dried under ultraviolet light for 10 min. Approximately 25 g peppers was inoculated with 10(8) to 10(1) colony forming units (CFU) of Salmonella enterica serovar Typhimurium in a stomacher bag and hand massaged in sterile 0.05 M glycine-0.14 M saline buffer (0.05% Tween, 3% beef extract) for optimal recovery of bacteria. A short preenrichment step of 6 h in buffered peptone water was needed for the detection of low inocula (10(4) CFU/25 g). One-milliliter portions of the extracts were serially diluted, plated on XLT4 agar, and used for RNA extraction with the Qiagen RNeasy Mini Kit. RT-PCR was carried out using SYBR Green I one-step RT-PCR with previously described invA gene primers and an internal amplification control. Detection limits were 10(4) CFU/25 g (approximately 10(2) CFU/g) and 10(7) CFU/25 g (approximately 10(5) CFU/g) Salmonella from enriched and unenriched inoculated peppers, respectively. Even though this method included a 6-h incubation period, the results were still obtainable in 1 day. This method shows promise for applications in routine surveillance and during outbreaks.

Topics: Bacterial Proteins; Bacterial Typing Techniques; Benzothiazoles; Capsicum; Cold Temperature; Colony Count, Microbial; Diamines; Electrophoresis, Agar Gel; Food Handling; Food Microbiology; Genes, Bacterial; Microchemistry; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Bacterial; Salmonella Food Poisoning; Salmonella typhimurium; Species Specificity; Stress, Physiological; Time Factors; Vegetables

A quantitative detection method for Salmonella in seafood was developed using a SYBR Green-based real-time PCR assay. The assay was developed using pure Salmonella DNA at different dilution levels [i.e., 1,000 to 2 genome equivalents (GE)]. The sensitivity of the real-time assay for Salmonella in seeded seafood samples was determined, and the minimum detection level was 20 CFU/g, whereas a detection level of 2 CFU/ml was obtained for pure culture in water with an efficiency of > or =85%. The real-time assay was evaluated in repeated experiments with seeded seafood samples and the regression coefficient (R(2)) values were calculated. The performance of the real-time assay was further assessed with naturally contaminated seafood samples, where 4 out of 9 seafood samples tested positive for Salmonella and harbored cells <100 GE/g, which were not detected by direct plating on Salmonella Chromagar media. Thus, the method developed here will be useful for the rapid quantification of Salmonella in seafood, as the assay can be completed within 2-3 h. In addition, with the ability to detect a low number of Salmonella cells in seafood, this proposed method can be used to generate quantitative data on Salmonella in seafood, facilitating the implementation of control measures for Salmonella contamination in seafood at harvest and post-harvest levels.

Topics: Animals; Bacterial Proteins; Benzothiazoles; Colony Count, Microbial; Diamines; DNA, Bacterial; Fishes; Food Microbiology; Organic Chemicals; Penaeidae; Polymerase Chain Reaction; Quinolines; Salmonella; Salmonella Food Poisoning; Seafood