Influence of primer sequences and DNA extraction method on detection of non-O157 shiga toxin-producing Escherichia coli in ground beef by real-time PCR targeting the eae, stx, and serogroup-specific genes
Wasilenko J.L.,U.S. Department of Agriculture |
Fratamico P.M.,U.S. Department of Agriculture |
Narang N.,50 College Station Road |
Tillman G.E.,50 College Station Road |
And 3 more authors.
Journal of Food Protection | Year: 2012
Non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, particularly those caused by the "big six" or "top six" non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Because of their significant public health impact and the notable prevalence of STEC in cattle, methods for detection of the big six non-O157 STEC in ground beef have been established. Currently, the U.S. Department of Agriculture, Food Safety and Inspection Service detection methods for screening beef samples for non-O157 STEC target the stx1, stx2, and eae virulence genes, with the 16S rRNA gene as an internal control, in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting genes in the E. coli O-antigen gene clusters of the big six non-O157 serogroups. The method that we previously reported was improved so that additional stx variants, stx1d, stx2e, and stx2g, are detected. Additionally, alignments of the primers targeting the eae gene were used to improve the detection assay so that eae subtypes that could potentially be of clinical significance would also be detected. Therefore, evaluation of alternative real-time PCR assay primers and probes for the stx and eae reactions was carried out in order to increase the stx and eae subtypes detected. Furthermore, a Tris-EDTA DNA extraction method was compared with a previously used procedure that was based on a commercially available reagent. The Tris-EDTA DNA extraction method significantly decreased the cycle threshold values for the stx assay (P < 0.0001) and eae assay (P < 0.0001), thereby increasing the ability to detect the targets. The use of different stx primers and probes increased the subtypes detected to include stx1d, stx2e, and stx2g, and sequence data showed that modification of the eae primer should allow the known eae subtypes to be detected. Copyright ©, International Association for Food Protection.
Hodges C.B.,Georgia Southern University |
Kim C.M.,University of Georgia |
Kim C.M.,50 College Station Road
TechTrends | Year: 2013
This study was conducted to investigate the effectiveness of a treatment designed to improve college algebra students' attitudes toward mathematics. Keller's ARCS motivational design model was used as a guiding framework for the development of a motivational video, which was delivered online. The application of motivational design to improve mathematics attitudes in an online environment extends the use of motivational design. A pretest-posttest control group design was used to test the effectiveness of the treatment. The participants in this study were 43 students enrolled in a college algebra course offered at a large state university in the mid-Atlantic region of the United States. Statistically significant results were observed for improved attitudes toward mathematics. © 2013 Association for Educational Communications and Technology.