Rapid and Simultaneous Quantitation of Escherichia coli O157:H7, Salmonella, and Shigella in Ground Beef by Multiplex Real-Time PCR and Immunomagnetic Separation

Abstract

The objective of this study was to establish a multiplex real-time PCR for the simultaneous quantitation of Escherichia coli O157:H7, Salmonella, and Shigella. Genomic DNA for the real-time PCR was extracted by the boiling method. Three sets of primers and corresponding TaqMan probes were designed to target these three pathogenic bacteria. Multiplex real-time PCR was performed with TaqMan Universal PCR Master Mix in an ABI Prism 7700 Sequence Detection System. Final standard curves were calculated for each pathogen by plotting the threshold cycle value against the bacterial number (log CFU per milliliter) via linear regression. With optimized conditions, the quantitative detection range of the real-time multiplex PCR for pure cultures was 10(2) to 10(9) CFU/ml for E. coli O157:H7, 10(3) to 10(9) CFU/ml for Salmonella, and 10(1) to 10(8) CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 10(5) CFU/g for E. coli O157:H7, 10(3) CFU/g for Salmonella, and 10(4) CFU/g for Shigella. Immunomagnetic separation (IMS) was further used to separate E. coli O157:H7 and Salmonella from the beef samples. With the additional use of IMS, the detection limit was 10(3) CFU/g for both pathogens. Results from this study showed that TaqMan real-time PCR, combined with IMS, is potentially an effective method for the rapid and reliable quantitation of E. coli 0157:H7, Salmonella, and Shigella in food.

Development of a Cell Culture Method To Isolate and Enrich Salmonella enterica Serotype Enteritidis from Shell Eggs for Subsequent Detection by Real-Time PCR

“…These methods are frequently being replaced with more-rapid and -sensitive molecular biology-based techniques. RT-PCR is a recent advance in molecular detection that has been used successfully for the rapid identification of a wide array of bacterial pathogens in specific food matrices (12,15,22,34,36,40). However, various food products have been shown to interfere with PCR assays, resulting in potentially false-negative results (1,22,31,32,33,35).…”

“…However, these methods require lengthy enrichment steps prior to the application of the respective methods. Real-time PCR (RT-PCR) is a promising new method currently used for detection of a wide variety of bacterial pathogens in food matrices (12,15,22,34,40). However, this technique can be ineffective for the detection of Salmonella serotype Enteritidis in foods such as eggs due to the presence of PCR-inhibitory components (41).…”

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.…”

Current and Emerging Innovations for Detection of Food-Borne Salmonella

“…It was reported that only one food item was positive for Salmonella [57]. Wang et al reported a qPCR method to detect Salmonella in raw sausage meat with detection limit of 4 cfu/g [58]. He also used this method to quantify Salmonella detection limits of 2.5 cfu/25 g for salmon and minced meat, 5 cfu/25 g of chicken meat, and 5 cfu/25 ml for raw milk, respectively [59].…”