Loop-Mediated Isothermal Amplification Assays for Detecting Shiga Toxin-Producing Escherichia coli in Ground Beef and Human Stools

Abstract

Shiga toxin-producing Escherichia coli (STEC), encompassing E. coli O157 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 linked to severe human illnesses such as hemorrhagic colitis and hemolytic-uremic syndrome. In this study, the stx 1 , stx 2 , and eae genes were chosen as targets to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, sensitive, and quantitative detection of STEC strains. The assay performances in pure culture and spiked ground beef and human stools were evaluated and compared with those of quantitative PCR (qPCR). No falsepositive or false-negative results were observed among 90 bacterial strains used to evaluate assay specificity. The limits of detection for seven STEC strains of various serogroups (O26, O45, O103, O111, O121, O145, and O157) were approximately 1 to 20 CFU/reaction in pure culture and 10 3 to 10 4 CFU/g in spiked ground beef, which were comparable to the results of 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 to 2 and 10 to 20 CFU/25 g) of STEC cultures, the LAMP assays achieved accurate detection after 6 to 8 h enrichment. The assays also consistently detected STEC in human stool specimens spiked with 10 3 or 10 4 CFU/0.5 g stool after 4 h enrichment, while qPCR required 4 to 6 h. In conclusion, the LAMP assays developed in this study may facilitate rapid and reliable identification of STEC contaminations in high-risk food commodities and also facilitate prompt diagnosis of STEC infections in clinical laboratories.

Comparison of Conventional PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Arcobacter Species

“…With DNA isolated from cultured bacteria, LAMP could detect as little as 2 CFU per reaction. This value is similar to those reported in previous studies, which detected 1, 20, 7.9, and 3.8 CFU per reaction for Escherichia coli O157:H7, Plesiomonas shigelloides, C. jejuni, and C. coli, respectively (30,31,40). The sensitivity of the LAMP method developed in this study was 10-to 1,000-fold higher than that of conventional PCR or multiplex PCR (30,40).…”

“…Bst DNA polymerase has a very high activity; thus, vast amounts of high-molecularweight DNA can be produced within a short time (28), with the reaction proceeding at a constant temperature. LAMP was first introduced in 2000, and it has been successfully used for the detection of many pathogens (28)(29)(30)(31).…”

Rapid and Specific Detection of Escherichia coli Serogroups O26, O45, O103, O111, O121, O145, and O157 in Ground Beef, Beef Trim, and Produce by Loop-Mediated Isothermal Amplification

“…A total of 91 E. coli strains of 20 serogroups (Table 1) and 29 non-E. coli strains as described in our recent study (47) were used for specificity testing. The E. coli serogroup information was taken directly from the source database (Table 1).…”

“…LAMP is also advantageous over PCR in that positive results can be directly detected through visual observation of turbidity changes (34). We recently developed and evaluated a set of serogroup-independent STEC LAMP assays by targeting common STEC virulence genes (stx 1 , stx 2 , and eae), and the assays were rapid, sensitive, specific, and robust with ground beef and human stool samples (47). Additionally, several STEC O157 LAMP assays targeting the rfbE gene (encoding perosamine synthetase) have been reported (46,52,53).…”

Usability and Performance of CHROMagar STEC Medium in Detection of Shiga Toxin-Producing Escherichia coli Strains

“…Therefore, rapid and reliable detection of STEC isolates in food and clinical samples is required. The direct molecular detection of Shiga toxin (Stx1 or Stx2) genes alone or in combination with other genes coding for major STEC virulence factors by nucleic acid-based techniques is known to be highly sensitive and specific in STEC detection (1,2,10,25). Major disadvantages of these assays are, however, the labor-intensive sample preparation and the increase of the costs when used only for screening purposes.…”