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1.
FEMS Microbiol Lett ; 262(2): 185-92, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16923074

ABSTRACT

We describe a real-time multiplexed PCR method using Taqman probes for the detection of total and pandemic Vibrio parahaemolyticus O3:K6 serovar in oysters and Gulf of Mexico water (gulf water). The specificity of these primers and probes was tested for amplification of a 450 bp thermolabile hemolysin (tlh) and a 369 bp ORF8 amplicon representing all V. parahaemolyticus and post-1996 clinical isolates of pandemic serovar O3:K6, respectively. The sensitivity of detection was 10 pg purified DNA or 10(3) CFU in 1 mL pure culture. Enrichment of this pathogen in oyster tissue homogenate or gulf water for 5 or 8 h resulted in the detection of an initial inoculum of 1 CFU in 1 mL or 1 g of samples. Application of the Taqman PCR assay on natural oysters exhibited a positive detection of V. parahaemolyticus, ranging from 16% to 100% of the samples collected primarily during the summer months. None of the samples exhibited a positive detection of O3:K6 serovar. Rapid and sensitive detection of this pathogen will help shellfish industry and Interstate Shellfish Sanitation Conference (ISSC) undertake appropriate measures to monitor this pathogen in oysters and oyster-growing waters, thereby preventing disease outbreaks and consequently protecting consumer health.


Subject(s)
Ostreidae/microbiology , Polymerase Chain Reaction/methods , Seawater/microbiology , Shellfish/microbiology , Vibrio Infections/epidemiology , Vibrio parahaemolyticus/isolation & purification , Animals , Atlantic Ocean , DNA Probes , Disease Outbreaks , Gastroenteritis/epidemiology , Gastroenteritis/microbiology , Humans , Sensitivity and Specificity , Serotyping , Vibrio Infections/microbiology , Vibrio parahaemolyticus/classification , Vibrio parahaemolyticus/genetics
2.
Appl Environ Microbiol ; 70(1): 498-507, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14711681

ABSTRACT

In this paper we describe optimization of SYBR Green I-based real-time PCR parameters and testing of a large number of microbial species with vvh-specific oligonucleotide primers to establish a rapid, specific, and sensitive method for detection of Vibrio vulnificus in oyster tissue homogenate and Gulf of Mexico water (gulf water). Selected oligonucleotide primers for the vvh gene were tested for PCR amplification of a 205-bp DNA fragment with a melting temperature of approximately 87 degrees C for 84 clinical and environmental strains of V. vulnificus. No amplification was observed with other vibrios or nonvibrio strains with these primers. The minimum level of detection by the real-time PCR method was 1 pg of purified genomic DNA or 10(2) V. vulnificus cells in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. It was possible to improve the level of detection to one V. vulnificus cell in samples that were enriched for 5 h. The standard curves prepared from the real-time PCR cycle threshold values revealed that there was a strong correlation between the number of cells in unenriched samples and the number of cells in enriched samples. Detection of a single cell of V. vulnificus in 1 g of enriched oyster tissue homogenate is in compliance with the recent Interstate Shellfish Sanitation Conference guidelines. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. Rapid and sensitive detection of V. vulnificus would ensure a steady supply of postharvest treated oysters to consumers, which should help decrease the number of illnesses or outbreaks caused by this pathogen.


Subject(s)
Ostreidae/microbiology , Polymerase Chain Reaction/methods , Seawater/microbiology , Shellfish/microbiology , Vibrio vulnificus/isolation & purification , Animals , Benzothiazoles , DNA, Bacterial/analysis , Diamines , Fluorescent Dyes , Hemolysin Proteins/genetics , Humans , Organic Chemicals , Quinolines , Sensitivity and Specificity , Time Factors , Vibrio vulnificus/genetics
3.
Appl Environ Microbiol ; 69(4): 2194-200, 2003 Apr.
Article in English | MEDLINE | ID: mdl-12676700

ABSTRACT

This study describes the optimization of PCR parameters and testing of a wide number of microbial species to establish a highly specific and sensitive PCR-based method of detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 strain in pure cultures and seeded waters from the Gulf of Mexico (gulf water). The selected open reading frame 8 (ORF8) DNA-specific oligonucleotide primers tested were found to specifically amplify all 35 pathogenic V. parahaemolyticus O3:K6 pandemic isolates, whereas these primers were not found to detectably amplify two strains of V. parahaemolyticus O3:K6 that were isolated prior to the 1996 outbreaks, 122 non-O3:K6 strains of V. parahaemolyticus, 198 non-V. parahaemolyticus spp., or 16 non-Vibrio bacterial spp. The minimum level of detection by the PCR method was 1 pg of purified genomic DNA or 10(2) ORF8-positive V. parahaemolyticus O3:K6 cells in 100 ml of water. The effectiveness of this method for the detection of ORF8-positive isolates in environmental samples was tested in gulf water seeded with 10-fold serial dilutions of this pathogen. A detection level of 10(3) cells per 100 ml of gulf water was achieved. Also, the applicability of this methodology was tested by the detection of this pathogen in gulf water incubated at various temperatures for 28 days. This PCR approach can potentially be used to monitor with high specificity and well within the required range of sensitivity the occurrence and distribution of this newly emerged pathogenic V. parahaemolyticus O3:K6 strain in coastal, marine, and ship ballast waters. Early detection of V. parahaemolyticus O3:K6 will help increase seafood safety and decrease the risk of infectious outbreaks caused by this pathogen.


Subject(s)
Communicable Diseases, Emerging/epidemiology , Disease Outbreaks , Polymerase Chain Reaction/methods , Seawater/microbiology , Vibrio parahaemolyticus/isolation & purification , Bacterial Typing Techniques , Communicable Diseases, Emerging/microbiology , Culture Media , DNA Primers , Humans , Open Reading Frames/genetics , Sensitivity and Specificity , Sequence Analysis, DNA , Vibrio Infections/epidemiology , Vibrio Infections/microbiology , Vibrio parahaemolyticus/classification , Vibrio parahaemolyticus/genetics , Vibrio parahaemolyticus/pathogenicity
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