Detection of Shiga Toxin-Producing Escherichia coli Using Loop Mediated Isothermal Amplification

Richelle J Allen
2013
Dedication: I dedicate this thesis to my father, Tim Allen. You were the hardest working; most amazing person I know and you were taken too soon. You inspired me to always push harder and take that next step, even when it wasn't easy. I took a path in life you may not have understood but you always supported me 100% and for that I will be forever grateful. Abstract: Shiga toxin-producing Escherichia coli (STEC) is a worldwide health concern. To detect STEC, a loop mediated isothermal
more » ... othermal amplification reaction (LAMP) was optimized to detect shiga toxin genes. LAMP's performance was compared to conventional and real-time (RT) PCR using two product detection methods. All three DNA amplification methods produced similar results. LAMP performed well when tested with randomly selected stool samples. LAMP, with agarose gel detection, showed a sensitivity of 90%/100%, specificity of 95%/99%, a positive predictive value of 56%/89% and a negative predictive value of 99%/100% for stx1 and stx2 respectively. Sybr Green 1 detection had a sensitivity of 100%/100%, specificity of 93%/77%, positive predictive values of 76/39% and negative predictive values of 100/100% for stx1 and stx2 respectively. Per 10 tests LAMP costs approximately $45, when using a rapid lysis DNA extraction and agarose gel electrophoresis product detection. LAMP could be implemented in laboratories without dedicated molecular biology facilities. Acknowledgements:
doi:10.7939/r3w08wp6k fatcat:iyt7lspfarcixppjvxkotrwdeu