Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays

Gitika Panicker; Douglas R Call; Melissa J Krug; Asim K Bej

doi:10.1128/AEM.70.12.7436-7444.2004

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Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays

Journal article

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Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays

Gitika Panicker, Douglas R Call, Melissa J Krug and Asim K Bej

Applied and environmental microbiology, Vol.70(12), pp.7436-7444

12/2004

DOI: https://doi.org/10.1128/AEM.70.12.7436-7444.2004

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https://hdl.handle.net/2376/115625

PMCID: PMC535186

PMID: 15574946

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https://doi.org/10.1128/AEM.70.12.7436-7444.2004View

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Abstract

Vibrio parahaemolyticus - isolation & purification

Vibrio vulnificus - classification

Vibrio vulnificus - genetics

Oligonucleotide Array Sequence Analysis - methods

Vibrio - genetics

Vibrio cholerae - genetics

Vibrio parahaemolyticus - classification

Humans

Vibrio cholerae - classification

Vibrio - classification

Vibrio - isolation & purification

Polymerase Chain Reaction - methods

Vibrio parahaemolyticus - genetics

Vibrio vulnificus - isolation & purification

Animals

Sensitivity and Specificity

Shellfish - microbiology

Vibrio cholerae - isolation & purification

Vibrio - pathogenicity

Ostreidae - microbiology

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 degrees 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 10(2) to 10(3) 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.

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Title: Detection of pathogenic Vibrio spp. in shellfish by using multiplex PCR and DNA microarrays
Creators: Gitika Panicker - Department of Biology, University of Alabama at Birmingham, 1300 University Blvd., Birmingham, AL 35294-1170, USA
Douglas R Call
Melissa J Krug
Asim K Bej
Publication Details: Applied and environmental microbiology, Vol.70(12), pp.7436-7444
Academic Unit: Paul G. Allen School for Global Animal Health
Publisher: United States
Identifiers: 99900547448601842
Language: English
Resource Type: Journal article