Rapid Enzymatic Detection of Shiga-Toxin-Producing E. coli Using Fluorescence-Labeled Oligonucleotide Substrates

被引：0

作者：

Ramming, Isabell ^{[1
]}

Lang, Christina ^{[1
]}

Hauf, Samuel ^{[1
]}

Krueger, Maren ^{[2
]}

Worbs, Sylvia ^{[2
]}

Peukert, Carsten ^{[3
]}

Fruth, Angelika ^{[1
]}

Dorner, Brigitte G. ^{[2
]}

Broenstrup, Mark ^{[3
,4
]}

Flieger, Antje ^{[1
]}

机构：

[1] Robert Koch Inst, Natl Reference Ctr Salmonella & Other Enter Bacter, Dept Infect Dis, Div Enteropathogen Bacteria & Legionella FG11, D-38855 Wernigerode, Germany

[2] Robert Koch Inst, Ctr Biol Threats & Special Pathogens, Biol Toxins ZBS3, D-13353 Berlin, Germany

[3] Helmholtz Ctr Infect Res, Dept Chem Biol CBIO, D-38124 Braunschweig, Germany

[4] German Ctr Infect Res DZIF, Site Hannover Braunschweig, D-38124 Braunschweig, Germany

来源：

ACS INFECTIOUS DISEASES | 2024年 / 10卷 / 12期

关键词：

Shiga-toxin-producing Escherichia coli detection; Shigatoxin; N-glycosidase; sarcin ricinloop; FRET; ssDNA SRL substrates; ESCHERICHIA-COLI; RIBOSOME DEPURINATION; ELISA MICROARRAY; ASSAY; STRAINS; QUANTIFICATION; PCR;

D O I：

10.1021/acsinfecdis.4c00221

中图分类号：

R914 [药物化学];

学科分类号：

100701 ;

摘要：

Shiga-toxin-producing Escherichia coli (STEC) are important human pathogens causing diarrhea, hemorrhagic colitis, and severe hemolytic uremic syndrome. Timely detection of the multifaceted STEC is of high importance but is challenging and labor-intensive. An easy-to-perform rapid test would be a tremendous advance. Here, the major STEC virulence factor Shiga toxins (Stx), RNA-N-glycosidases targeting the sarcin ricin loop (SRL) of 28S rRNA, was used for detection. We designed synthetic FRET-based ssDNA SRL substrates, which conferred a fluorescence signal after cleavage by Stx. Optimal results using bacterial culture supernatants or single colonies were achieved for substrate StxSense 4 following 30 to 60 min incubation. Stx1 and Stx2 subtypes, diverse STEC serotypes, and Shigella were detected. Within a proof-of-principle study, a total of 94 clinical strains were tested, comprising 65 STEC, 11 Shigella strains, and 18 strains of other enteropathogenic bacteria without Stx. In conclusion, the assay offers rapid and facile STEC detection based on a real-time readout for Stx activity. Therefore, it may improve STEC risk evaluation, therapy decisions, outbreak, and source detection and simplify research for antimicrobials.

引用

页码：4103 / 4114

页数：12

共 41 条

[21] Comparison of eight different agars for the recovery of clinically relevant non-O157 Shiga toxin-producing Escherichia coli from baby spinach, cilantro, alfalfa sprouts and raw milk [J].