Quantitative detection of Escherichia coli O157:H7 in ground beef by the polymerase chain reaction incorporating immunomagnetic separation
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.
School Location:USA - Massachusetts
Source Type:Master's Thesis
Date of Publication:01/01/2002