Development of DNA-tagged liposome biosensing devices and enzyme-linked dot blot assay in combination with nucleic acid sequence-based amplification for rapid detection of viable shiga toxin-producing Escherichia coli.

摘要

Shiga toxin-producing Escherichia coli (STEC) are a group of newly emerging food-borne pathogens with a very low infectious dose. Currently, the main detection methods focus principally on E. coli O157:H7, the most notorious member of this group. However, reliance on a test which detects only the O157:H7 serotype, but misses the other STEC strains, would put public health at risk. This research was to develop novel assay systems based on the combination of nucleic acid sequence-based amplification (NASBA) with enzyme-linked dot blot assay, or DNA-tagged, dye-encapsulating liposomes for the specific identification of viable STEC.; Mitomycin C was included in the culture medium for Shiga toxin (Stx) induction, and chloramphenicol was used for pre-harvest treatment to improve the recovery of intact mRNAs from cells. One set of primers derived from a conserved region among stx mRNA sequences was applied in target amplification using NASBA. For enzyme-linked dot blot assay, the detection signal was generated through horseradish peroxidase activity. As for the capillary-migration, sandwich hybridization dual strip assay, two capture probe-immobilized strips, each specific to Stx1- or Stx2-producing strains, were developed to capture the amplified RNA products by using two distinct reporter probetagged, dye-loaded liposomes as the sensing devices. To further simplify the dual strip assay by immobilizing Stx1- and Stx2-specific capture probes in two different positions on one strip, and performing two sandwich hybridization reactions in one setting, the liposome single strip assay system was created. All assay systems developed here were able to differentiate Stx1- from Stx2-producing E. coli in all tested STEC protypes. The detection limit of these assays with pure cultures of STEC was 1 CFU/ml. After 5 h of selective enrichment, liposome-based strip assays allowed the detection of STEC spiked in ground beef at a level equivalent to or below 1 CFU/g. The whole assay procedure, including enrichment, could be completed in less than 9 h. The strip assay systems are easy to perform, and all the components of the assays can be prepared in advance and stored. By using mRNA as the target, these detection systems are rapid and sensitive methods for detection of viable STEC in foods.