Detection and Categorization of Diarrheagenic Escherichia coli with Auto-microfluidic Thin-film Chip Method

Abstract

Diarrheagenic Escherichia coli (DEC) causes human diarrhea symptom in both healthy and immunocompromised individuals. An auto-microfluidic thin-film chip (AMTC) instrument integrating one-step multiplex PCR (mPCR) with reverse dot blot hybridization (RDBH) was developed for high-throughput detection of DEC. The novel mPCR method was developed by designing 14 specific primers and corresponding probes. 14 indexes including an endogenous gene (uidA) and 13 pathogenic genes (stx1, stx2, escV, ipaH, invE, estB, lt, pic, aggR, astA, bfpB, sth and stp) of DEC were detected. This one-step mPCR + RDBH approach is useful for simultaneous detection of numerous target genes in a single sample, whose specificity and availability have been confirmed on the positive control of 11 DEC strains. In addition, with 300 diarrheal stool samples being detected by this method, 21 were found to contain five major DEC strains. Compared with monoplex PCR and previous one-step mPCR approach, this method could detect ipaH and estB, and compared with current commercial kit, the relevance ratio of DEC detected by the AMTC method was increased by 1% in stool samples. Furthermore, the novel integration AMTC device could be a valuable detection tool for categorization of E. coli.

Figure 1

Design of microfluidic thin-film chip. (A) Overview of microfluidic thin-film chip. (B) Arrangement of DEC probes on the thin-film-based array. uidA, β-glucuronidase gene; stx (stx1, stx2), shiga-like toxin I (stx1), shiga-like toxin II (stx2); escV, gene encoding LEE (locus of enterocyte effacement)-encoded type III secretion system factor; ipaH, invasive plasmid antigen H-gene; invE, invasive plasmid regulator; estB, heat-stable enterotoxin b; lt, heat-labile enterotoxin; bfpB, bundle-forming pilus B; pic, protein involved in intestinal colonization; aggR, aggregative adhesive fimbriae regulator; astA, enteroaggregative heat-stable enterotoxin A; sth, heat-stable enterotoxins initially discovered in the isolates from human; stp, heat-stable enterotoxins initially discovered in the isolates from pigs; PC, positive control; and NC, negative control.

Figure 2

Electrophoretograms of monoplex PCR for DEC strains. BP01 includes uidA, stx1, stx2, escV and astA, being identified as EHEC; BP02 includes uidA and ipaH, identified as EIEC; BP03 includes uidA, ipaH, invE and pic, identified as EIEC; BP04 includes uidA and escV, identified as atypical EPEC; BP05 includes uidA, escV and bfpB, identified as typical EPEC; BP06 and BP07 include uidA, but no virulence genes; BP08 includes uidA, lt and stp, identified as ETEC; BP09 includes uidA, lt, astA and sth, identified as ETEC; BP10 includes uidA, pic and aggR, identified as EAEC; and BP11 includes uidA, pic, aggR and astA, identified as EAEC.

Figure 3

Electrophoretogram of one-step mPCR for DEC strains. BP01 includes uidA, stx1, stx2, escV and astA, being identified as EHEC; BP02 includes uidA, but no virulence genes; BP03 includes uidA, invE and pic, identified as EIEC; BP04 includes uidA and escV, identified as atypical EPEC; BP05 includes uidA, escV and bfpB, identified as typical EPEC; BP06 and BP07 include uidA, but no virulence genes; BP08 includes uidA, lt and stp, identified as ETEC; BP09 includes uidA, lt, astA and sth, identified as ETEC; BP10 includes uidA, pic and aggR, identified as EAEC; and BP11 includes uidA, pic, aggR and astA, identified as EAEC.

Figure 4

Qualitative detection of DEC strains. BP01 includes uidA, stx1, stx2, escV and astA, being identified as EHEC; BP02 includes uidA and ipaH, identified as EIEC; BP03 includes uidA, ipaH, invE and pic, identified as EIEC; BP04 includes uidA and escV, identified as atypical EPEC; BP05 includes uidA, escV and bfpB, identified as typical EPEC; BP06 includes uidA and estB, identified as ETEC; BP07 includes uidA, but no virulence genes; BP08 includes uidA, lt and stp, identified as ETEC; BP09 includes uidA, lt, astA and sth, identified as ETEC; BP10 includes uidA, pic and aggR, identified as EAEC; BP11 includes uidA, pic, aggR and astA, identified as EAEC.

Figure 5

Electrophoretogram of one-step mPCR for diarrheal stool samples. YP11 includes uidA, stx1, stx2 and escV, being identified as EHEC; YP55 and YP60 include uidA and invE, identified as EIEC; YP74, YP90, YP108, YP166 and YP299 include uidA and escV, identified as atypical EPEC;YP179 includes uidA, escV and bfpB, identified as typical EPEC; YP186 and YP191 include uidA, lt and stp, identified as ETEC; YP192 includes uidA, lt and sth, identified as ETEC; YP222 includes uidA and sth, identified as ETEC; YP223,YP248 and YP295 include uidA, but no virulence genes; YP255 and YP293 include uidA and pic, identified as EAEC; and YP276, YP277 and YP280 include uidA, pic and aggR, identified as EAEC.

Figure 6

Qualitative detection of DEC strains in diarrheal stool samples by RDBH on AMTC instrument.YP11 includes uidA, stx1, stx2 and escV, being identified as EHEC; YP55 and YP60 include uidA, ipaH and invE, identified as EIEC; YP74 and YP90 include uidA and escV, identified as atypical EPEC; YP108 and YP166 include uidA, astA and escV, identified as atypical EPEC;YP179 includes uidA, escV and bfpB, identified as typical EPEC; YP186 includes uidA, lt and stp, identified as ETEC; YP191 includes uidA, astA, lt and stp, identified as ETEC; YP192 includes uidA, lt, astA and sth, identified as ETEC;YP222 includes uidA, astA and sth, identified as ETEC;YP223 includes uidA and estB, identified as ETEC; YP248 includes uidA, astA and estB, identified as ETEC; YP255 and YP293 include uidA, astA and pic, identified as EAEC; YP276, YP277 and YP280 include uidA, pic, aggR and astA, identified as EAEC; YP295 includes uidA and astA, but no virulence genes; and YP299 includes uidA, estB and escV, identified as atypical EPEC and ETEC.