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Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef.
J Microbiol Methods. 2004 Nov; 59(2):243-52.JM

Abstract

In this study, enrichment procedures and two recovery methods, a membrane surface adhesion technique and an immunomagnetic separation (IMS), were compared for use in conjunction with a multiplex polymerase chain reaction (PCR) method with a view to describing a fast (24 h) and economical test for detection of Escherichia coli O157:H7 in meat samples. The study showed no significant difference between three different enrichment media (BHI, E. coli (E.C.) broth+novobiocin, modified tryptone soya broth (mTSB)+novobiocin) or two incubation temperatures (37 or 41.5 degrees C) for growth of E. coli O157:H7 in minced beef. Minced beef samples inoculated with E. coli O157:H7 at 40 cfu g(-1) were incubated at 37 degrees C for 16 h in E.C. broth+novobiocin reaching numbers of (log(10)7.82-8.70). E. coli O157:H7 were recovered by attachment to polycarbonate membranes immersed in the enriched cultures for 15 min or by immunomagnetic separation. Subsequent treatment of recovered membranes or IMS beads with lysis buffer and phenol/chloroform/isoamyl alcohol was used to extract the DNA from the extracted E. coli O157:H7 cells. The results show when E. coli O157:H7 was present at high levels in the enriched meat sample (log(10)9.6-7.5 cfu ml(-1); >16-h enrichment), the membrane and IMS techniques recovered similar levels of the pathogen and the microorganism was detectable by PCR using both methods. At lower levels of E. coli O157:H7 (log(10)6.4), only the IMS method could recover the pathogen but at levels below this neither method could recover sufficient numbers of the pathogens to allow detection. The conclusion of the study is that with sufficient enrichment time (16 h) the membrane surface adhesion membrane extraction method used in combination with multiplex PCR has the potential for a rapid and economical detection method.

Authors+Show Affiliations

Fitzmaurice J
The National Diagnostics Centre, BioResearch Ireland, National University of Ireland, Galway, Ireland.
Duffy G
No affiliation info available
Kilbride B
No affiliation info available
Sheridan JJ
No affiliation info available
Carroll C
No affiliation info available
Maher M
No affiliation info available

MeSH

AnimalsBacterial AdhesionCattleDNA, BacterialEscherichia coli O157Food MicrobiologyImmunomagnetic SeparationMeatPolymerase Chain ReactionPolymers

Pub Type(s)

Comparative Study
Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

15369860

Citation

Fitzmaurice, J, et al. "Comparison of a Membrane Surface Adhesion Recovery Method With an IMS Method for Use in a Polymerase Chain Reaction Method to Detect Escherichia Coli O157:H7 in Minced Beef." Journal of Microbiological Methods, vol. 59, no. 2, 2004, pp. 243-52.
Fitzmaurice J, Duffy G, Kilbride B, et al. Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef. J Microbiol Methods. 2004;59(2):243-52.
Fitzmaurice, J., Duffy, G., Kilbride, B., Sheridan, J. J., Carroll, C., & Maher, M. (2004). Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef. Journal of Microbiological Methods, 59(2), 243-52.
Fitzmaurice J, et al. Comparison of a Membrane Surface Adhesion Recovery Method With an IMS Method for Use in a Polymerase Chain Reaction Method to Detect Escherichia Coli O157:H7 in Minced Beef. J Microbiol Methods. 2004;59(2):243-52. PubMed PMID: 15369860.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Comparison of a membrane surface adhesion recovery method with an IMS method for use in a polymerase chain reaction method to detect Escherichia coli O157:H7 in minced beef. AU - Fitzmaurice,J, AU - Duffy,G, AU - Kilbride,B, AU - Sheridan,J J, AU - Carroll,C, AU - Maher,M, PY - 2003/07/23/received PY - 2004/07/12/revised PY - 2004/07/13/accepted PY - 2004/9/17/pubmed PY - 2004/12/16/medline PY - 2004/9/17/entrez SP - 243 EP - 52 JF - Journal of microbiological methods JO - J Microbiol Methods VL - 59 IS - 2 N2 - In this study, enrichment procedures and two recovery methods, a membrane surface adhesion technique and an immunomagnetic separation (IMS), were compared for use in conjunction with a multiplex polymerase chain reaction (PCR) method with a view to describing a fast (24 h) and economical test for detection of Escherichia coli O157:H7 in meat samples. The study showed no significant difference between three different enrichment media (BHI, E. coli (E.C.) broth+novobiocin, modified tryptone soya broth (mTSB)+novobiocin) or two incubation temperatures (37 or 41.5 degrees C) for growth of E. coli O157:H7 in minced beef. Minced beef samples inoculated with E. coli O157:H7 at 40 cfu g(-1) were incubated at 37 degrees C for 16 h in E.C. broth+novobiocin reaching numbers of (log(10)7.82-8.70). E. coli O157:H7 were recovered by attachment to polycarbonate membranes immersed in the enriched cultures for 15 min or by immunomagnetic separation. Subsequent treatment of recovered membranes or IMS beads with lysis buffer and phenol/chloroform/isoamyl alcohol was used to extract the DNA from the extracted E. coli O157:H7 cells. The results show when E. coli O157:H7 was present at high levels in the enriched meat sample (log(10)9.6-7.5 cfu ml(-1); >16-h enrichment), the membrane and IMS techniques recovered similar levels of the pathogen and the microorganism was detectable by PCR using both methods. At lower levels of E. coli O157:H7 (log(10)6.4), only the IMS method could recover the pathogen but at levels below this neither method could recover sufficient numbers of the pathogens to allow detection. The conclusion of the study is that with sufficient enrichment time (16 h) the membrane surface adhesion membrane extraction method used in combination with multiplex PCR has the potential for a rapid and economical detection method. SN - 0167-7012 UR - https://www.unboundmedicine.com/medline/citation/15369860/Comparison_of_a_membrane_surface_adhesion_recovery_method_with_an_IMS_method_for_use_in_a_polymerase_chain_reaction_method_to_detect_Escherichia_coli_O157:H7_in_minced_beef_ DB - PRIME DP - Unbound Medicine ER -