Development of methodology for quantitative detection of E. coli O157:H7 in ground beef via immunoassays and the polymerase chain reaction
Abstract (Summary)Escherichia coli O157:H7 is recognized as a major human enteropathogen. Immunotechnology and DNA based methods were developed for quantitative detection of E. coli O157:H7 in ground beef. A direct spectrophotometric immuno-agglutination assay was developed for quantitation of specific Escherichia coli O157 IgG. Optimum conditions of the assay consisted of 1 Ã?-- 108 cells/ml, 40Ã?Â°C, and 0.005M phosphate buffer containing 0.05% NaCl at pH 7.4. The assay was able to quantitate (13 to 104) Ã?Â¼g IgG/ml of anti O157 IgG in crude antiserum and was effectively used with different batches of locally produced antisera. A new cell lysis solution designated TZ (2.0% Triton X-100 in 0.1 M Tris-HCl buffer plus 2.5 mg sodium azide/ml, pH 8.0) was developed. TZ lysis solution was found superior to a variety of cell lysing methods (d.H2 O, PCR buffer, SDS, Triton X-100, proteinase K, and lysozyme in combination with proteinase K) and released the greatest yield of E. coli O157:H7 DNA targets prior to PCR. Highest amplification of E. coli O157:H7 SLT-1 and SLT-2 DNA sequences were achieved with the aid of TZ lysis solution and pellet paint. Using 0.01 M phosphate buffered saline pH 6.0 with the aid of differential centrifugation resulted in 57% Ã?Â± 6 recovery of E. coli O157:H7 from seeded ground beef. The optimization of PCR reaction mixture resulted in a minimum detection level of 100 SLT-1 DNA sequence compared to 200 DNA targets before the optimization. Prior to optimization, the minimum limits for the SLT-2 DNA sequence was 150 DNA targets compared to 20 after complete optimization. The complete optimization of PCR reaction mixture contained 1 mM MgCl2 , 1.6 Ã?Â¼M each of the two primers, (2.5 and 5 units) of Taq polymerase for SLT-1 and SLT-2 respectively, and 0.2 mM of Deoxynucleotide Triphosphates (dNTPs). The methodologies developed thereby resulted in the detection of 100 cells of E. coli O157:H7 with SLT-1 primer and 50 cells with SLT-2 primer per 10 grams of ground beef after 5.5 hours of pre-enrichment in 30 ml of TSB+ at 37Ã?Â°C.
School Location:USA - Massachusetts
Source Type:Master's Thesis
Date of Publication:01/01/2006