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Optimization and Application of a Custom Microarray for the Detection and Genotyping of E. coli O157:H7 in Fresh Meat Samples

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Abstract

DNA microarrays are promising high-throughput tools for multiple pathogen detection. Currently, the performance and cost of this platform has limited its broad application in identifying microbial contaminants in foods. In this study, an optimized custom DNA microarray with flexibility in design and content for foodborne pathogen detection was developed through the systematic evaluation of spotting buffers, probe lengths, scanning conditions, and nucleic acid amplification and labeling strategies. Briefly, by robotic contact printing, a spotting solution of 50 % dimethylsulfoxide produced uniform and high-quality spots on UltraGAPS glass slides coated with aminopropyl silane. The use of 60 % photomultiplier tube gain in scanning ∼70-mer oligonucleotide probes resulted in strong signals and low background. For sample preparation, multiplex PCR amplification coupled with fluorescent labeling of DNA using the Klenow fragment and random hexamers achieved higher specificity than whole genome random amplification. To minimize the cost of the assay, the quantities of probes, Klenow fragment, and Cy5 were substantially reduced in each assay without noticeably affecting the detection efficiency. Applying the optimized microarray assay to 26 fresh meat samples, three different isolates of Escherichia coli O157:H7 were found in four individual packages, demonstrating that the assay has a great potential for identifying and genotyping multiple pathogens in a real food system.

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References

  • Barbulovic-Nad I, Lucente M, Sun Y, Zhang MJ, Wheeler AR, Bussmann M (2006) Bio-microarray fabrication techniques—a review. Crit Rev Biotechnol 26:237–259

    Article  CAS  Google Scholar 

  • Battaglia C, Salani G, Consolandi C, Bernardi LR, De Bellis G (2000) Analysis of DNA microarrays by non-destructive fluorescent staining using SYBR green II. Biotechniques 29:78–81

    CAS  Google Scholar 

  • Bilge SS, Vary J, Dowell SF, Tarr PI (1996) Role of the Escherichia coli O157:H7 O side chain in adherence and analysis of an rfb locus. Infect Immun 64:4795–4801

    CAS  Google Scholar 

  • Chen W, Yu S, Zhang C, Zhang J, Shi C, Hu Y, Suo B, Cao H, Shi X (2010) Development of a single base extension-tag microarray for the detection of pathogenic Vibrio species in seafood. Appl Microbiol Biotechnol 89:1979–1990

    Article  Google Scholar 

  • Chudin E, Walker R, Kosaka A, Wu SX, Rabert D, Chang TK, Kreder DE (2002) Assessment of the relationship between signal intensities and transcript concentration for Affymetrix GeneChip arrays. Genome Biol 3:0005.0001–0005.0010

    Google Scholar 

  • Dawson ED, Reppert AE, Rowlen KL, Kuck LR (2005) Spotting optimization for oligo microarrays on aldehyde-glass. Anal Biochem 341:352–360

    Article  CAS  Google Scholar 

  • Gould LH, Demma L, Jones TF, Hurd S, Vugia DJ, Smith K, Shiferaw B, Segler S, Palmer A, Zansky S (2009) Hemolytic uremic syndrome and death in persons with Escherichia coli O157:H7 infection, foodborne diseases active surveillance network sites, 2000–2006. Clin Infect Dis 49:1480–1485

    Article  Google Scholar 

  • Hussein HS (2007) Prevalence and pathogenicity od Shiga toxin-producing Escherichia coli in beef cattle and their products. J Anim Sci 85:E63–E72

    Article  CAS  Google Scholar 

  • Kim H, Bhunia AK (2008) SEL, a selective enrichment broth for simultaneous growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. Appl Environ Microbiol 74:4853–4866

    Article  CAS  Google Scholar 

  • Kostic T, Stessl B, Wagner M, Sessitsch A (2011) Microarray analysis reveals the actual specificity of enrichment media used for food safety assessment. J Food Prot 74:1030–1034

    Article  Google Scholar 

  • Louie M, de Azavedo JC, Handelsman MY, Clark CG, Ally B, Dytoc M, Sherman P, Brunton J (1993) Expression and characterization of the eaeA gene product of Escherichia coli serotype O157:H7. Infect Immun 61:4085–4092

    CAS  Google Scholar 

  • Manduchi E, Scearce LM, Brestelli JE, Grant GR, Kaestner KH, Stoeckert CJ (2002) Comparison of different labeling methods for two-channel high-density microarray experiments. Physiol Genom 10:169–179

    CAS  Google Scholar 

  • Nugen SR, Baeumner AJ (2008) Trends and opportunities in food pathogen detection. Anal Bioanal Chem 391:451–454

    Article  CAS  Google Scholar 

  • O’Brien AD, Holmes RK (1987) Shiga and Shiga-like toxins. Microbiol Rev 51:206–220

    Google Scholar 

  • Palka-Santini M, Cleven BE, Eichinger L, Krönke M, Krut O (2009) Large scale multiplex PCR improves pathogen detection by DNA microarrays. BMC Microbiol 9:1

    Article  Google Scholar 

  • Reid SD, Selander RK, Whittam TS (1999) Sequence diversity of flagellin (fliC) alleles in pathogenic Escherichia coli. J Bacteriol 181:153–160

    CAS  Google Scholar 

  • Schmidt H, Plaschke B, Franke S, Rüssmann H, Schwarzkopf A, Heesemann J, Karch H (1994) Differentiation in virulence patterns of Escherichia coli possessing eae genes. Med Microbiol Immun 183:23–31

    Article  CAS  Google Scholar 

  • Severgnini M, Cremonesi P, Consolandi C, De Bellis G, Castiglioni B (2011) Advances in DNA microarray technology for the detection of foodborne pathogens. Food Biop Technol 4:936–953

    Article  Google Scholar 

  • Shi XM, Long F, Suo B (2010) Molecular methods for the detection and characterization of foodborne pathogens. Pure Appl Chem 82:69–79

    Article  CAS  Google Scholar 

  • Suo B, He Y, Paoli G, Gehring A, Tu SI, Shi X (2010a) Development of an oligonucleotide-based microarray to detect multiple foodborne pathogens. Mol Cell Probes 24:77–86

    Article  CAS  Google Scholar 

  • Suo B, He Y, Tu SI, Shi X (2010b) A multiplex real-time polymerase chain reaction for simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes in meat products. Foodborne Pathog Dis 7:619–628

    Article  CAS  Google Scholar 

  • Uda A, Tanabayashi K, Fujita O, Hotta A, Yamamoto Y, Yamada A (2007) Comparison of whole genome amplification methods for detecting pathogenic bacterial genomic DNA using microarray. Jpn J Infect Dis 60:355–361

    CAS  Google Scholar 

  • Uhlich GA, Sinclair JR, Warren NG, Chmielecki WA, Fratamico P (2008) Characterization of Shiga toxin-producing Escherichia coli isolates associated with two multistate food-borne outbreaks that occurred in 2006. Appl Environ Microbiol 74:1268–1272

    Article  CAS  Google Scholar 

  • Wang XW, Zhang L, Jin LQ, Jin M, Shen ZQ, An S, Chao FH, Li JW (2007) Development and application of an oligonucleotide microarray for the detection of food-borne bacterial pathogens. Appl Microbiol Biotechnol 76:225–233

    Article  CAS  Google Scholar 

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Acknowledgments

We would like to acknowledge the US Department of Agriculture, Agricultural Research Service (USDA-ARS), the National Natural Science Foundation of China (grant No. U1204331), and the Science and Technology Department of Henan Province (grant No. 122102310310) for supporting this research. Mention of trade names or commercial products is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. The USDA is an equal opportunity provider and employer.

Conflict of Interest

Biao Suo declare no conflict of interest. Yiping He declare no conflict of interest. Peter Irwin declare no conflict of interest. Andrew Gehring declare no conflict of interest. This article does not contain any studies with human or animal subjects.

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Authors and Affiliations

  1. College of Food Science and Technology, Henan Agricultural University, Zhengzhou, 450002, China

    Biao Suo

  2. Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center, Agricultural Research Service, United States Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA, 19038, USA

    Yiping He, Peter Irwin & Andrew Gehring

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  1. Biao Suo
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  2. Yiping He
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  4. Andrew Gehring
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Correspondence to Yiping He.

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Suo, B., He, Y., Irwin, P. et al. Optimization and Application of a Custom Microarray for the Detection and Genotyping of E. coli O157:H7 in Fresh Meat Samples. Food Anal. Methods 6, 1477–1484 (2013). https://doi.org/10.1007/s12161-013-9639-z

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  • DOI: https://doi.org/10.1007/s12161-013-9639-z

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