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Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR.
Int J Food Microbiol. 2005 Mar 01; 99(1):47-57.IJ

Abstract

A method combining immunomagnetic separation (IMS) and real-time (5'-nuclease) PCR was developed to detect Escherichia coli O157:H7. Monoclonal antibody specific for the E. coli O157 antigen was added to protein A-coated magnetic particles to create antibody-coated beads. The beads specifically captured E. coli O157:H7 from bacterial suspensions. The cells were eluted from the beads and lysed by heating; the eluate was then assayed by real-time PCR, using primers and probe specifically targeting the eaeA gene of E. coli O157:H7. Approximately 50% of the cells in suspension were captured by the beads and detected by real-time PCR. No cross-reactivity was detected when other strains of E. coli were tested. This method was applied to detect E. coli O157:H7 from ground beef. Both cell capture efficiency and real-time PCR efficiency were reduced by meat-associated inhibitors. However, we were still able to detect up to 8% of E. coli O157:H7 from inoculated ground beef samples. The detection sensitivity varied among ground beef samples. The minimum detection limit was <5x10(2) cells ml(-1) for suspensions of E. coli O157:H7 in buffer and 1.3x10(4) cells g(-1) for E. coli O157:H7 in ground beef. The combination of IMS and real-time PCR results in rapid, specific and quantitative detection of E. coli O157:H7 without the need for an enrichment culture step.

Authors+Show Affiliations

Fu Z
Biology Department, New Mexico Institute of Mining and Technology, Socorro, NM 87801, USA.
Rogelj S
No affiliation info available
Kieft TL
No affiliation info available

MeSH

Adhesins, BacterialAnimalsAntibodies, MonoclonalCattleColony Count, MicrobialCross ReactionsEscherichia coli O157Escherichia coli ProteinsFood ContaminationImmunomagnetic SeparationMeat ProductsPolymerase Chain ReactionSensitivity and Specificity

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

15718028

Citation

Fu, Zhu, et al. "Rapid Detection of Escherichia Coli O157:H7 By Immunomagnetic Separation and Real-time PCR." International Journal of Food Microbiology, vol. 99, no. 1, 2005, pp. 47-57.
Fu Z, Rogelj S, Kieft TL. Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR. Int J Food Microbiol. 2005;99(1):47-57.
Fu, Z., Rogelj, S., & Kieft, T. L. (2005). Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR. International Journal of Food Microbiology, 99(1), 47-57.
Fu Z, Rogelj S, Kieft TL. Rapid Detection of Escherichia Coli O157:H7 By Immunomagnetic Separation and Real-time PCR. Int J Food Microbiol. 2005 Mar 1;99(1):47-57. PubMed PMID: 15718028.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Rapid detection of Escherichia coli O157:H7 by immunomagnetic separation and real-time PCR. AU - Fu,Zhu, AU - Rogelj,Snezna, AU - Kieft,Thomas L, PY - 2003/08/15/received PY - 2004/04/20/revised PY - 2004/07/26/accepted PY - 2005/2/19/pubmed PY - 2005/4/9/medline PY - 2005/2/19/entrez SP - 47 EP - 57 JF - International journal of food microbiology JO - Int J Food Microbiol VL - 99 IS - 1 N2 - A method combining immunomagnetic separation (IMS) and real-time (5'-nuclease) PCR was developed to detect Escherichia coli O157:H7. Monoclonal antibody specific for the E. coli O157 antigen was added to protein A-coated magnetic particles to create antibody-coated beads. The beads specifically captured E. coli O157:H7 from bacterial suspensions. The cells were eluted from the beads and lysed by heating; the eluate was then assayed by real-time PCR, using primers and probe specifically targeting the eaeA gene of E. coli O157:H7. Approximately 50% of the cells in suspension were captured by the beads and detected by real-time PCR. No cross-reactivity was detected when other strains of E. coli were tested. This method was applied to detect E. coli O157:H7 from ground beef. Both cell capture efficiency and real-time PCR efficiency were reduced by meat-associated inhibitors. However, we were still able to detect up to 8% of E. coli O157:H7 from inoculated ground beef samples. The detection sensitivity varied among ground beef samples. The minimum detection limit was <5x10(2) cells ml(-1) for suspensions of E. coli O157:H7 in buffer and 1.3x10(4) cells g(-1) for E. coli O157:H7 in ground beef. The combination of IMS and real-time PCR results in rapid, specific and quantitative detection of E. coli O157:H7 without the need for an enrichment culture step. SN - 0168-1605 UR - https://www.unboundmedicine.com/medline/citation/15718028/Rapid_detection_of_Escherichia_coli_O157:H7_by_immunomagnetic_separation_and_real_time_PCR_ DB - PRIME DP - Unbound Medicine ER -