Real-time reverse-transcriptase--polymerase chain reaction for Salmonella enterica detection from jalapeño and serrano peppers.
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.
Miller
Nathan D
ND
Department of Food Science and Technology, The University of Tennessee, Knoxville, Tennessee, USA.
Draughon
Frances Ann
FA
D'Souza
Doris H
DH
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015373:Bacterial Typing Techniques / Q000191:economics / Q000379:methods; D052160:Benzothiazoles; D002212:Capsicum / Q000382:microbiology*; D003080:Cold Temperature / Q000009:adverse effects; D015169:Colony Count, Microbial; D003959:Diamines; D004587:Electrophoresis, Agar Gel; D005511:Food Handling / Q000191:economics / Q000379:methods; D005516:Food Microbiology; D005798:Genes, Bacterial; D008832:Microchemistry / Q000379:methods; D009930:Organic Chemicals; D011804:Quinolines; D012329:RNA, Bacterial / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012478:Salmonella Food Poisoning / Q000517:prevention & control; D012486:Salmonella typhimurium / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D013045:Species Specificity; D013312:Stress, Physiological; D013997:Time Factors; D014675:Vegetables / Q000382:microbiology*
D001426:Bacterial Proteins; D052160:Benzothiazoles; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D012329:RNA, Bacterial; C075518:invA protein, Bacteria; C098022:SYBR Green I