Detection of pandemic Vibrio parahaemolyticus O3:K6 serovar in Gulf of Mexico water and shellfish using real-time PCR with Taqman fluorescent probes.
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.
Rizvi
Amy V
AV
Department of Biology, University of Alabama at Birmingham, Birmingham, AL 35294-1170, USA.
Panicker
Gitika
G
Myers
Michael L
ML
Bej
Asim K
AK
D000818:Animals; D001267:Atlantic Ocean; D015342:DNA Probes; D004196:Disease Outbreaks; D005759:Gastroenteritis / Q000453:epidemiology / Q000382:microbiology; D006801:Humans; D010125:Ostreidae / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012623:Seawater / Q000382:microbiology*; D012680:Sensitivity and Specificity; D012703:Serotyping; D012758:Shellfish / Q000382:microbiology*; D014735:Vibrio Infections / Q000453:epidemiology* / Q000382:microbiology; D014736:Vibrio parahaemolyticus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
D015342:DNA Probes