Abstract/Details

Quantitative detection of Escherichia coli O157:H7 in ground beef by the polymerase chain reaction incorporating immunomagnetic separation


2002 2002

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Abstract (summary)

Escherichia coli O157:H7 is an important foodborne pathogen. The majority of outbreaks of this organism have been attributed to ground beef. In order to improve the sensitivity for detection of E. coli O157:H7 in ground beef by the polymerase chain reaction (PCR), sample preparation methods including differential centrifugation, enrichment, immunomagnetic separation (IMS), and DNA extraction were studied. Quantitative detection protocols based on standard PCR, nested PCR and competitive PCR were developed.

Using standard PCR enabled detection of 150 colony forming units (CFU) per gram of ground beef without enrichment and 1.2 CFU/g after 4.5 h of enrichment in modified EC broth plus novobiocin (mEC+n) with SLT I and SLT II primers respectively. The quantitative detection range was 150 to 15,000 CFU/g for protocol without enrichment and 1.2 to 19.2 CFU/g for protocol with enrichment.

Standard PCR incorporating IMS detected 70 CFU/g with SLT I and SLT II primers without enrichment and 0.7 CFU/g after 6 h of enrichment in Tryptic Soy Broth (TSB) and IMS. The quantitative detection range was 70 to 24,000 CFU/g for protocol without enrichment and 0.7 to 70 CFU/g for protocol with enrichment. A partial digestion of the cell pellets derived from differential centrifugation was required for IMS. DNA extracted from seeded target cells captured by IMB could be used for PCR amplification without further purification.

Nested PCR incorporating differential centrifugation and IMS was capable of detecting 2.4 CFU/g with SLT 1 and SLT 2 primers without enrichment. Within the range of 2.4 to 240 CFU/g, a linear relationship between the log CFU of the inocula and the fluorescent intensity of secondary PCR products was achieved by using 25 cycles for both primary and secondary PCR run.

Competitive PCR incorporating IMS enabled quantitative detection of 0.5 to 5.5 CFU per gram of ground beef with SLT I and SLT II primers after 6 hours of enrichment using TSB. The numbers of target organisms after enrichment and IMS determined by competitive PCR were highly correlated to those derived from viable plate counts on violet red bile agar.

Indexing (details)


Subject
Food science
Classification
0359: Food science
Identifier / keyword
Biological sciences, Escherichia coli O157:H7
Title
Quantitative detection of Escherichia coli O157:H7 in ground beef by the polymerase chain reaction incorporating immunomagnetic separation
Author
Guan, Jiewen
Number of pages
121
Publication year
2002
Degree date
2002
School code
0118
Source
DAI-B 63/01, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
ISBN
9780493525822, 0493525823
Advisor
Levin, Robert E.
University/institution
University of Massachusetts Amherst
University location
United States -- Massachusetts
Degree
Ph.D.
Source type
Dissertations & Theses
Language
English
Document type
Dissertation/Thesis
Dissertation/thesis number
3039361
ProQuest document ID
251662566
Copyright
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
http://search.proquest.com/docview/251662566
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