Simultaneous direct detection of Shiga-toxin producing Escherichia coli (STEC) strains by optical biosensing with oligonucleotide-functionalized gold nanoparticles.

A simultaneous direct detection of Shiga-toxin producing strains of E. coli (STEC; "Big Six" - O26, O45, O103, O111, O121, and O145) as well as O157 strains by optical biosensing with oligonucleotide-functionalized gold nanoparticles (AuNPs) was developed. Initially, conserved regions of stx genes were amplified by asymmetric polymerase chain reaction (asPCR). Pairs of single stranded thiol-modified oligonucleotides (30-mer) were immobilized onto AuNPs and used as probes to capture regions of stx1 (119-bp) and/or stx2 (104-bp) genes from STEC strains. DNA samples from pure cultures and food samples were sandwich hybridized with AuNP-oligo probes at optimal conditions (50 °C, 30 min). A complex was formed from the hybridization of AuNP-probes and target DNA fragments that retained the initial red color of the reaction solutions. For non-target DNA, a color change from red to purplish-blue was observed following an increase in salt concentration, thus providing the basis of simultaneous direct colorimetric detection of target DNA in the samples. Enrichment and pooling systems were incorporated to efficiently process a large number of food samples (ground beef and blueberries) and detection of live targets. The detection limit was <1 log CFU g(-1), requiring less than 1 h to complete after DNA sample preparation with 100% specificity. Gel electrophoresis verified AuNP-DNA hybridization while spectrophotometric data and transmission electron microscope (TEM) images supported color discrimination based on the occurrence of molecular aggregation. In conclusion, the significant features of this approach took advantage of the unique colorimetric properties of AuNPs as a low-cost and simple approach yet with high specificity for simultaneous detection of STEC strains.

[1]  W. Prinyawiwatkul,et al.  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 , 2012, Applied and Environmental Microbiology.

[2]  Yanbin Li,et al.  Simultaneous detection of Escherichia coli O157:H7 and Salmonella Typhimurium using quantum dots as fluorescence labels. , 2006, The Analyst.

[3]  J. Blanco,et al.  Serotypes, Virulence Genes, and Intimin Types of Shiga Toxin (Verotoxin)-Producing Escherichia coli Isolates from Human Patients: Prevalence in Lugo, Spain, from 1992 through 1999 , 2004, Journal of Clinical Microbiology.

[4]  J. Wells,et al.  Non-O157 Shiga toxin-producing Escherichia coli infections in the United States, 1983-2002. , 2005, The Journal of infectious diseases.

[5]  J. Paton,et al.  Direct Detection and Characterization of Shiga Toxigenic Escherichia coli by Multiplex PCR for stx1, stx2, eae, ehxA, and saa , 2002, Journal of Clinical Microbiology.

[6]  Yingfu Li,et al.  DNA aptamer folding on gold nanoparticles: from colloid chemistry to biosensors. , 2008, Journal of the American Chemical Society.

[7]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[8]  K. Ariga,et al.  Gold Nanoparticles Aggregation: Drastic Effect of Cooperative Functionalities in a Single Molecular Conjugate , 2012 .

[9]  George C Schatz,et al.  What controls the melting properties of DNA-linked gold nanoparticle assemblies? , 2000, Journal of the American Chemical Society.

[10]  Sarit S. Agasti,et al.  Gold nanoparticles in chemical and biological sensing. , 2012, Chemical reviews.

[11]  A. Gill,et al.  Development of a method for the detection of verotoxin-producing Escherichia coli in food. , 2012, Journal of food protection.

[12]  Bowen Zhu,et al.  Optical reading of contaminants in aqueous media based on gold nanoparticles. , 2014, Small.

[13]  C. Tang,et al.  Optical Detection of Human Papillomavirus Type 16 and Type 18 by Sequence Sandwich Hybridization With Oligonucleotide-Functionalized Au Nanoparticles , 2009, IEEE Transactions on NanoBioscience.

[14]  Olivier Lazcka,et al.  Pathogen detection: a perspective of traditional methods and biosensors. , 2007, Biosensors & bioelectronics.

[15]  Michael A. Brook,et al.  Design of Gold Nanoparticle‐Based Colorimetric Biosensing Assays , 2008, Chembiochem : a European journal of chemical biology.

[16]  Miss A.O. Penney.  (b) , 1974, The New Yale Book of Quotations.

[17]  Andrew G. Glen,et al.  APPL , 2001 .

[18]  Hilde Kruse,et al.  Food-borne diseases — The challenges of 20 years ago still persist while new ones continue to emerge , 2010, International Journal of Food Microbiology.

[19]  Mieke Uyttendaele,et al.  Alternative microbial methods: An overview and selection criteria. , 2010, Food microbiology.

[20]  S. Shackelford,et al.  Evaluation of commonly used antimicrobial interventions for fresh beef inoculated with Shiga toxin-producing Escherichia coli serotypes O26, O45, O103, O111, O121, O145, and O157:H7. , 2012, Journal of food protection.

[21]  Yingfu Li,et al.  Simple and Rapid Colorimetric Biosensors Based on DNA Aptamer and Noncrosslinking Gold Nanoparticle Aggregation , 2007, Chembiochem : a European journal of chemical biology.

[22]  H. Karch,et al.  Real-Time Fluorescence PCR Assays for Detection and Characterization of Shiga Toxin, Intimin, and Enterohemolysin Genes from Shiga Toxin-Producing Escherichia coli , 2002, Journal of Clinical Microbiology.

[23]  Y. Hara-Kudo,et al.  Rapid detection of Shiga toxin-producing Escherichia coli in ground beef by an immunochromatography kit in combination with short-term enrichment and treatment for Shiga toxin release. , 2011, Biocontrol science.

[24]  Chih-Sheng Lin,et al.  Real-time detection of Escherichia coli O157:H7 sequences using a circulating-flow system of quartz crystal microbalance. , 2007, Biosensors & bioelectronics.

[25]  Fan Yang,et al.  Applications of Gold Nanoparticles in the Detection and Identification of Infectious Diseases and Biothreats , 2013, Advanced materials.

[26]  Khalil Arshak,et al.  An overview of foodborne pathogen detection: in the perspective of biosensors. , 2010, Biotechnology advances.

[27]  A. Valadez,et al.  Detection of Shiga toxin-producing Escherichia coli O26, O45, O103, O111, O113, O121, O145, and O157 serogroups by multiplex polymerase chain reaction of the wzx gene of the O-antigen gene cluster. , 2011, Foodborne pathogens and disease.

[28]  D. Bolton.  Verocytotoxigenic (Shiga toxin-producing) Escherichia coli: virulence factors and pathogenicity in the farm to fork paradigm. , 2011, Foodborne pathogens and disease.

[29]  Chitra N. Wendakoon,et al.  Detection of E. coli O157:H7 in raw ground beef by Pathatrix™ immunomagnetic-separation, real-time PCR and cultural methods. , 2011, International journal of food microbiology.

[30]  P. Fratamico,et al.  Isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 from ground beef using modified rainbow agar and post-immunomagnetic separation acid treatment. , 2012, Journal of food protection.

[31]  B. Liedberg,et al.  Polypeptide folding-mediated tuning of the optical and structural properties of gold nanoparticle assemblies. , 2011, Nano letters.

[32]  Robin Patel,et al.  Rapid and Sensitive Detection of Shiga Toxin-Producing Escherichia coli from Nonenriched Stool Specimens by Real-Time PCR in Comparison to Enzyme Immunoassay and Culture , 2009, Journal of Clinical Microbiology.

[33]  S. Derzelle,et al.  A quantitative PCR assay for the detection and quantification of Shiga toxin-producing Escherichia coli (STEC) in minced beef and dairy products. , 2011, International journal of food microbiology.