Purpose: The objective of this study was to develop a method to determine the contamination of various Salmonella spp. strains in poultry, produce and environmental samples by employing an efficient pooling and immunomagnetic separation (IMS) system coupled with rapid optical biosensing detection using functionalized-gold nanoparticles (AuNPs).
Methods: Pairs of single stranded thiol-modified oligonucleotides (30-mer) were immobilized onto AuNPs and used as probes to capture ttrRSBCA region (192-bp) from asymmetric polymerase chain reaction (asPCR). DNA samples from pure cultures, inoculated chicken and blueberries, and natural environmental samples were sandwich hybridized with AuNP-oligo probes at optimal conditions (55oC, 40 minutes). A complex was formed from the hybridized AuNP-probes and target DNA fragment which allowed retention of the initial red color of the target reaction solutions following an increase in salt concentration. For non-target DNA, a color change from red to purplish-blue was observed. Shortened enrichment (6 hours) and pooling of samples using Pathatrix IMS system were utilized to ensure detection of viable cells.
Results: From a total of 55 individual samples (chicken = 25; blueberry = 25; environment = 5), 40 samples tested positive for Salmonella spp. All environmental samples tested negative. The assay had a superior detection limit of 1 log CFU/g with 100% specificity and required less than one hour to complete after DNA sample preparation. Transmission electron microscope (TEM) images confirmed AuNP-DNA hybridization while spectrophotometric data supported the color discrimination based on the occurrence of molecular aggregation.
Significance: The significant features of this study took advantage of the unique colorimetric properties of AuNPs, shortened enrichment with robust sampling and pooling systems for rapid detection of Salmonella spp. even at a low contamination level.