Journal article
Simultaneous Detection of Marine Fish Pathogens by Using Multiplex PCR and a DNA Microarray
Journal of clinical microbiology, Vol.42(4), pp.1414-1419
04/2004
Handle:
https://hdl.handle.net/2376/105604
PMCID: PMC387578
PMID: 15070982
Abstract
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.
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Details
- Title
- Simultaneous Detection of Marine Fish Pathogens by Using Multiplex PCR and a DNA Microarray
- Creators
- Santiago F González - Department of Microbiology and Parasitology, University of Santiago de Compostela, 15706 Santiago de Compostela, SpainMelissa J Krug - Department of Microbiology and Parasitology, University of Santiago de Compostela, 15706 Santiago de Compostela, SpainMichael E Nielsen - Department of Microbiology and Parasitology, University of Santiago de Compostela, 15706 Santiago de Compostela, SpainYsabel Santos - Department of Microbiology and Parasitology, University of Santiago de Compostela, 15706 Santiago de Compostela, SpainDouglas R Call - Department of Microbiology and Parasitology, University of Santiago de Compostela, 15706 Santiago de Compostela, Spain
- Publication Details
- Journal of clinical microbiology, Vol.42(4), pp.1414-1419
- Academic Unit
- Paul G. Allen School for Global Animal Health
- Publisher
- American Society for Microbiology
- Identifiers
- 99900547076101842
- Language
- English
- Resource Type
- Journal article