This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Panicker, G.
Right arrow Articles by Bej, A. K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Panicker, G.
Right arrow Articles by Bej, A. K.
Agricola
Right arrow Articles by Panicker, G.
Right arrow Articles by Bej, A. K.

 Previous Article  |  Next Article 

Applied and Environmental Microbiology, December 2004, p. 7436-7444, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7436-7444.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Detection of Pathogenic Vibrio spp. in Shellfish by Using Multiplex PCR and DNA Microarrays

Gitika Panicker,1 Douglas R. Call,2 Melissa J. Krug,2 and Asim K. Bej1*

Department of Biology, University of Alabama at Birmingham, Birmingham, Alabama,1 Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington2

Received 10 May 2004/ Accepted 26 July 2004

This study describes the development of a gene-specific DNA microarray coupled with multiplex PCR for the comprehensive detection of pathogenic vibrios that are natural inhabitants of warm coastal waters and shellfish. Multiplex PCR with vvh and viuB for Vibrio vulnificus, with ompU, toxR, tcpI, and hlyA for V. cholerae, and with tlh, tdh, trh, and open reading frame 8 for V. parahaemolyticus helped to ensure that total and pathogenic strains, including subtypes of the three Vibrio spp., could be detected and discriminated. For DNA microarrays, oligonucleotide probes for these targeted genes were deposited onto epoxysilane-derivatized, 12-well, Teflon-masked slides by using a MicroGrid II arrayer. Amplified PCR products were hybridized to arrays at 50°C and detected by using tyramide signal amplification with Alexa Fluor 546 fluorescent dye. Slides were imaged by using an arrayWoRx scanner. The detection sensitivity for pure cultures without enrichment was 102 to 103 CFU/ml, and the specificity was 100%. However, 5 h of sample enrichment followed by DNA extraction with Instagene matrix and multiplex PCR with microarray hybridization resulted in the detection of 1 CFU in 1 g of oyster tissue homogenate. Thus, enrichment of the bacterial pathogens permitted higher sensitivity in compliance with the Interstate Shellfish Sanitation Conference guideline. Application of the DNA microarray methodology to natural oysters revealed the presence of V. vulnificus (100%) and V. parahaemolyticus (83%). However, V. cholerae was not detected in natural oysters. An assay involving a combination of multiplex PCR and DNA microarray hybridization would help to ensure rapid and accurate detection of pathogenic vibrios in shellfish, thereby improving the microbiological safety of shellfish for consumers.

* Corresponding author. Mailing address: Department of Biology, University of Alabama at Birmingham, 1300 University Blvd., Birmingham, AL 35294-1170. Phone: (205) 934-9857. Fax: (205) 975-6097. E-mail: abej{at}uab.edu.

Applied and Environmental Microbiology, December 2004, p. 7436-7444, Vol. 70, No. 12
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.12.7436-7444.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jin, S.-Q., Yin, B.-C., Ye, B.-C. (2009). Multiplexed Bead-Based Mesofluidic System for Detection of Food-Borne Pathogenic Bacteria. Appl. Environ. Microbiol. 75: 6647-6654 [Abstract] [Full Text]  
  • Hazen, T. H., Martinez, R. J., Chen, Y., Lafon, P. C., Garrett, N. M., Parsons, M. B., Bopp, C. A., Sullards, M. C., Sobecky, P. A. (2009). Rapid Identification of Vibrio parahaemolyticus by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry. Appl. Environ. Microbiol. 75: 6745-6756 [Abstract] [Full Text]  
  • Li, Y., Cao, B., Liu, B., Liu, D., Gao, Q., Peng, X., Wu, J., Bastin, D. A., Feng, L., Wang, L. (2009). Molecular detection of all 34 distinct O-antigen forms of Shigella. J Med Microbiol 58: 69-81 [Abstract] [Full Text]  
  • Kim, H. S., Kim, D.-M., Neupane, G. P., Lee, Y.-m., Yang, N.-W., Jang, S. J., Jung, S.-I., Park, K.-H., Park, H.-R., Lee, C. S., Lee, S. H. (2008). Comparison of Conventional, Nested, and Real-Time PCR Assays for Rapid and Accurate Detection of Vibrio vulnificus. J. Clin. Microbiol. 46: 2992-2998 [Abstract] [Full Text]  
  • Miller, S. M., Tourlousse, D. M., Stedtfeld, R. D., Baushke, S. W., Herzog, A. B., Wick, L. M., Rouillard, J. M., Gulari, E., Tiedje, J. M., Hashsham, S. A. (2008). In Situ-Synthesized Virulence and Marker Gene Biochip for Detection of Bacterial Pathogens in Water. Appl. Environ. Microbiol. 74: 2200-2209 [Abstract] [Full Text]  
  • Nordstrom, J. L., Vickery, M. C. L., Blackstone, G. M., Murray, S. L., DePaola, A. (2007). Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters. Appl. Environ. Microbiol. 73: 5840-5847 [Abstract] [Full Text]  
  • Han, W., Liu, B., Cao, B., Beutin, L., Kruger, U., Liu, H., Li, Y., Liu, Y., Feng, L., Wang, L. (2007). DNA Microarray-Based Identification of Serogroups and Virulence Gene Patterns of Escherichia coli Isolates Associated with Porcine Postweaning Diarrhea and Edema Disease. Appl. Environ. Microbiol. 73: 4082-4088 [Abstract] [Full Text]  
  • Tarr, C. L., Patel, J. S., Puhr, N. D., Sowers, E. G., Bopp, C. A., Strockbine, N. A. (2007). Identification of Vibrio Isolates by a Multiplex PCR Assay and rpoB Sequence Determination. J. Clin. Microbiol. 45: 134-140 [Abstract] [Full Text]  
  • Tracz, D. M., Backhouse, P. G., Olson, A. B., McCrea, J. K., Walsh, J. A., Ng, L.-K., Gilmour, M. W. (2007). Rapid detection of Vibrio species using liquid microsphere arrays and real-time PCR targeting the ftsZ locus. J Med Microbiol 56: 56-65 [Abstract] [Full Text]  
  • Gubala, A. J., Proll, D. F. (2006). Molecular-Beacon Multiplex Real-Time PCR Assay for Detection of Vibrio cholerae. Appl. Environ. Microbiol. 72: 6424-6428 [Abstract] [Full Text]  
  • Vora, G. J., Meador, C. E., Bird, M. M., Bopp, C. A., Andreadis, J. D., Stenger, D. A. (2005). Microarray-based detection of genetic heterogeneity, antimicrobial resistance, and the viable but nonculturable state in human pathogenic Vibrio spp.. Proc. Natl. Acad. Sci. USA 102: 19109-19114 [Abstract] [Full Text]  
  • Bae, J.-W., Rhee, S.-K., Nam, Y.-D., Park, Y.-H. (2005). Generation of subspecies level-specific microbial diagnostic microarrays using genes amplified from subtractive suppression hybridization as microarray probes. Nucleic Acids Res 33: e113-e113 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»