Development and Application of Serogroup-independent and Serogroup-specific Loop-mediated Isothermal Amplification Assays for Detecting Shiga Toxin-Producing Escherichia coli

Abstract

Shiga toxin-producing Escherichia coli (STEC), encompassing E. coli O157:H7 and non-O157 STEC, is a significant cause of foodborne illnesses and deaths in the United States and worldwide. Shiga toxins (encoded by stx) and intimin (encoded by eae) are important virulence factors for STEC strains causing infection. Although E. coli O157:H7 remains to be the single most common STEC causing disease, the clinical importance of non-O157 STEC is on the rise worldwide. And six major serogroups (O26, O45, O103, O111, O121, and O145) accounted for over 70% of non-O157 STEC infections in the United States. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification technology that has attracted great attention in recent years as a rapid, accurate, and cost-effective pathogen detection method in both food testing and clinical diagnostics. In this dissertation research, two sets of LAMP assays, one serogroup-independent and the other one serogroup-specific, were designed by targeting the stx1, stx2, and eae genes, and seven major STEC serogroup-specific genes (the wzx and wzy genes), respectively, for the rapid, specific, sensitive, and quantitative detection of STEC strains. The assay performances in pure culture, spiked ground beef, and human stools were evaluated and compared with qPCR. No false positive or false negative results were observed among 120 strains for assay specificity testing. The detection limits for all assays were approximately 1-20 CFU/reaction in pure culture and 103-104 CFU/g in spiked ground beef, which were comparable to qPCR. Standard curves generated suggested good linear relationships between STEC cell numbers and LAMP turbidity signals. When applied in ground beef samples spiked with two low levels (1-2 and 10-20 CFU/25 g) of STEC cultures, the LAMP assays achieved accurate detection after 6-8 h of enrichment. The assays also consistently detected STEC in human stool specimens spiked with 103 or 104 CFU/0.5 g stool after 4 h enrichment, while qPCR required 4-6 h of enrichment. Given the emerging and evolving nature of STEC serogroups involved in human illness, the LAMP assays developed in this research can serve as rapid and reliable methods for STEC detection in food so that proper control measures can be implemented promptly.