Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR

The objective of this study was to develop a rapid, simple method for enhanced detection and isolation of low levels of Escherichia coli O157:H7 from leafy produce and surface water using recirculating immunomagnetic separation (RIMS) coupled with real-time PCR and a standard culture method. The optimal enrichment conditions for the method also were determined. Analysis of real-time PCR data (CT values) suggested that incubation of lettuce and spinach leaves rather than rinsates provides better enrichment of E. coli O157:H7. Enrichment of lettuce or spinach leaves at 42°C for 5 h provided better detection than enrichment at 37°C. Extended incubation of surface water for 20 h at 42°C did not improve the detection. The optimized enrichment conditions were also employed with modified Moore swabs, which were used to sample flowing water sites. Positive isolation rates and real-time PCR results indicated an increased recovery of E. coli O157:H7 from all samples following the application of RIMS. Under these conditions, the method provided detection and/or isolation of E. coli O157:H7 at levels as low as 0.07 CFU/g of lettuce, 0.1 CFU/g of spinach, 6 CFU/100 ml of surface water, and 9 CFU per modified Moore swab. During a 6-month field study, modified Moore swabs yielded high isolation rates when deployed in natural watershed sites. The method used in this study was effective for monitoring E. coli O157:H7 in the farm environment, during postharvest processing, and in foodborne outbreak investigations.

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Rapid detection for low numbers of Escherichia coli O157:H7 by real-time PCR in cabbage using a combination of filtration, short microbial enrichment, and DNA concentration within 4 h

LWT ◽

10.1016/j.lwt.2020.110520 ◽

2020 ◽

pp. 110520

Author(s):

Jin-Hee Kim ◽

Se-Wook Oh

Keyword(s):

Escherichia Coli ◽

Real Time ◽

Real Time Pcr ◽

Rapid Detection ◽

Escherichia Coli O157 ◽

Dna Concentration

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

Journal of Food Protection ◽

10.4315/0362-028x-70.6.1366 ◽

2007 ◽

Vol 70 (6) ◽

pp. 1366-1372 ◽

Cited By ~ 104

Author(s):

LUXIN WANG ◽

YONG LI ◽

AZLIN MUSTAPHA

Keyword(s):

Escherichia Coli ◽

Detection Limit ◽

Real Time ◽

Real Time Pcr ◽

Ground Beef ◽

Immunomagnetic Separation ◽

Quantitative Detection ◽

Escherichia Coli O157 ◽

The Real ◽

E Coli

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 102 to 109 CFU/ml for E. coli O157:H7, 103 to 109 CFU/ml for Salmonella, and 101 to 108 CFU/ml for Shigella. When the established multiplex real-time PCR system was applied to artificially contaminated ground beef, the detection limit was 105 CFU/g for E. coli O157:H7, 103 CFU/g for Salmonella, and 104 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 103 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 O157:H7, Salmonella, and Shigella in food.

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