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Journal of Clinical Microbiology, April 2004, p. 1414-1419, Vol. 42, No. 4
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.4.1414-1419.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Simultaneous Detection of Marine Fish Pathogens by Using Multiplex PCR and a DNA Microarray

Santiago F. González,1,2 Melissa J. Krug,3,4 Michael E. Nielsen,2 Ysabel Santos,1 and Douglas R. Call3,4*

Department of Microbiology and Parasitology, University of Santiago de Compostela, 15706 Santiago de Compostela, Spain,1 Department of Veterinary Microbiology and Pathology,3 WSU and UI Center for Reproductive Biology, Washington State University, Pullman, Washington,4 Section of Fish Diseases, Department of Veterinary Microbiology, Royal Veterinary and Agricultural University, DK-1870 Frederiksberg C, Denmark2

Received 27 August 2003/ Returned for modification 12 October 2003/ Accepted 18 December 2003

We coupled multiplex PCR and a DNA microarray to construct an assay suitable for the simultaneous detection of five important marine fish pathogens (Vibrio vulnificus, Listonella anguillarum, Photobacterium damselae subsp. damselae, Aeromonas salmonicida subsp. salmonicida, and Vibrio parahaemolyticus). The array was composed of nine short oligonucleotide probes (25-mer) complementary to seven chromosomal loci (cyt, rpoN, gyrB, toxR, ureC, dly, and vapA) and two plasmid-borne loci (fatA and A.sal). Nine primer sets were designed to amplify short fragments of these loci (100 to 177 bp) in a multiplex PCR. PCR products were subsequently labeled by nick translation and hybridized to the microarray. All strains of the five target species (n = 1 to 21) hybridized to at least one species-specific probe. Assay sensitivities ranged from 100% for seven probes to 83 and 67% for the two remaining probes. Multiplex PCR did not produce any nonspecific amplification products when tested against 23 related species of bacteria (n = 40 strains; 100% specificity). Using purified genomic DNA, we were able to detect PCR products with <20 fg of genomic DNA per reaction (equivalent to four or five cells), and the array was at least fourfold more sensitive than agarose gel electrophoresis for detecting PCR products. In addition, our method allowed the tentative identification of virulent strains of L. anguillarum serotype O1 based on the presence of the fatA gene (67% sensitivity and 100% specificity). This assay is a sensitive and specific tool for the simultaneous detection of multiple pathogenic bacteria that cause disease in fish and humans.

* Corresponding author. Mailing address: Department of Veterinary Microbiology and Pathology, Washington State University, 402 Bustad Hall, P.O. Box 647040, Pullman, WA 99164-7040. Phone: (509) 335-6313. Fax: (509) 335-8529. E-mail: drcall{at}wsu.edu.

Journal of Clinical Microbiology, April 2004, p. 1414-1419, Vol. 42, No. 4
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.4.1414-1419.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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