A Simple Method for the Direct Detection of Salmonella and Escherichia coli O157:H7 from Raw Alfalfa Sprouts and Spent Irrigation Water Using PCR

Abstract

The U.S. Food and Drug Administration recognizes that raw seed sprouts are an important cause of foodborne disease and is now recommending that either spent irrigation water or final product be screened for Salmonella and Escherichia coli O157:H7 as a means of assuring the safety of product intended for consumption. In an effort to streamline such testing efforts, a simple method to preconcentrate pathogens from sprouts and spent irrigation water was investigated to facilitate the direct (without prior cultural enrichment) detection of pathogens using the PCR technique. Alfalfa sprouts and spent irrigation water were seeded with Salmonella enterica serovar Typhimurium and E. coli O157:H7 at 10(-1) to 106 CFU/g or CFU/ml, respectively. Samples were blended (sprouts only) and then centrifuged at high speed to sediment the total bacterial population. The precipitate was processed for DNA isolation, PCR amplification, and amplicon confirmation by Southern hybridization. Mean pathogen recoveries after centrifugation ranged from 96 to 99% for both pathogens in both matrices. Using primers targeting the invA gene for Salmonella Typhimurium and the stx genes of E. coli O157:H7, it was possible to detect both pathogens in alfalfa sprouts at seeding concentrations as low as 10 CFU/g. PCR detection limits for both pathogens from spent irrigation water were 10(-1) CFU/ml, the equivalent of 100 CFU/liter of water. Because spent irrigation water is constitutionally simple, it is particularly well suited for bacterial concentration by simple centrifugation steps. In this study, progress was made toward development of a rapid, inexpensive, and sensitive method for the detection of sprout-associated pathogens that is relevant to current industrial practices and needs.

Sensing Escherichia coli O157:H7 via frequency shift through a self-assembled monolayer based QCM immunosensor

“…Some new methods have been developed, including immunoassays (Magliulo et al, 2007), immunomagnetic separations (Wang et al, 2007), nucleic acid probe-based methods based on hybridization and polymerase chain reaction (PCR) (Johnston et al, 2005), and the DNA microarrays (Jin et al, 2005). However, many of these methods are so time-consuming, expensive or complicated that they are not suitable for fast detection of E. coli O157:H7.…”

Flow cytometry for rapid detection of Salmonella spp. in seed sprouts

“…A number of approaches have been developed for more rapid testing of Salmonella in alfalfa or other types of sprouts (e.g. mung bean) and in SIW, including commercially available immunoassay [39] or custom amperometric immunoassay [40], standard PCR [41], automated fiber optic biosensor [42], or automated nucleic acid sensor [38]. Pre-analytical sample preparation is an indispensible element in successful navigation from sample to answer [43].…”

“…Sample preparation methods used in conjunction with assays for Salmonella in sprouts include simple centrifugation [41] or filtration [39] and TFF [40,44]. Reported levels of sensitivity for detection of Salmonella in SIW were 100 CFU L 1 (0.1 CFU ml 1 ) for standard PCR [41], ∼300 CFU ml 1 for combined TFF and amperometric immunoassay [40] and 8 Â 10 5 CFU ml 1 for automated PCR (reported as 10 4 CFU per 12.5 ml sample of SIW) [38]. These are all promising for rapid, and in the case of standard PCR, extremely sensitive detection-based interventions that might be leveraged for increasing the safety of seed sprouts.…”

Sample Preparation: The Forgotten Beginning

“…Although some sampling, separation, or concentration approaches may involve specialized instrumentation (e.g., wet vacuum-based surface sampling, recirculating immunocapture, continuous flow centrifugation), some samples may be amenable to the use of approaches much simpler and less capital intensive. Examples include the use of Scotch tape (or similar adhesive tapes) for sampling of produce, meats, or food contact surfaces (9,24); Kimwipe absorbent tissues for recovery of Listeria monocytogenes from stainless steel surfaces (86); and simple filtration or centrifugation steps for PCR-based detection of Salmonella or E. coli O157:H7 from chicken rinsate, alfalfa sprouts or mung bean, and sprout irrigation waters (32,93). Advantages of very simple methods for sampling or sample preparation include their relative ease to learn, apply, and troubleshoot, the wide availability of the raw materials or equipment needed to conduct the assay, speed (as a result of fewer and/or more rapidly accomplished steps), acceptable or improved efficacy or reproducibility vis-à-vis existing methods, and reduced expense on both per-assay and capital investment bases.…”