Rapid sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef

被引:43
作者
Cui, S
Schroeder, CM
Zhang, DY
Meng, J
机构
[1] Univ Maryland, Dept Nutr & Food Sci, College Pk, MD 20742 USA
[2] CUNY Mt Sinai Sch Med, Dept Pathol, New York, NY 10029 USA
关键词
detection; E. coli O157 : H7; ground beef; PCR;
D O I
10.1046/j.1365-2672.2003.01951.x
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Aim: To develop an improved, rapid and sensitive sample preparation method for PCR-based detection of Escherichia coli O157:H7 in ground beef. Methods and Results: Fresh ground beef samples were experimentally inoculated with varying concentrations of E. coli O157:H7. PCR inhibitors were removed and bacterial cells were concentrated by filtration and centrifugation, and lysed using enzymatic digestion and successive freeze/thaw cycles. DNA was purified and concentrated via phenol/chloroform extraction and the Shiga toxin 1 gene (stx1) was amplified using PCR to evaluate the sample preparation method. Without prior enrichment of cells in broth media, the detection limit was 10(3) CFU g(-1) beef. When a 6 h enrichment step was incorporated, the detection limit was 1 CFU g(-1) beef. The total time required from beginning to end of the procedure was 12 h. Conclusions: The sample preparation method developed here enabled substantially improved sensitivity in the PCR-based detection of E. coli O157:H7 in ground beef, as compared to previous reports. Significance and Impact of the Study: Superb sensitivity, coupled with quick turn-around time, relative ease of use and cost-effectiveness, makes this a useful method for detecting E. coli O157:H7 in ground beef.
引用
收藏
页码:129 / 134
页数:6
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