Real-Time PCR Detection of the Thermostable Direct Hemolysin and Thermolabile Hemolysin Genes in a Vibrio parahaemolyticus Cultured from Mussels and Mussel Homogenate Associated with a Foodborne Outbreak

Abstract

Molecular methods have become vital epidemiological tools in the detection and characterization of bacteria associated with a foodborne outbreak. We used both culture and real-time PCR to detect a Vibrio parahaemolyticus isolate associated with a foodborne outbreak. The outbreak occurred in July 2002 in Polk County, Florida, and originated at a Chinese buffet, with one person being hospitalized. The hospital isolated V. parahaemolyticus from the patient but destroyed the sample after diagnosis. From an onsite visit of the restaurant, food samples that possibly contributed to the outbreak were collected and sent to the Florida Department of Health, Tampa Branch Laboratory. Crab legs, crabsticks, and mussel samples were homogenized and incubated according to the Food and Drug Administration Bacteriological Analytical Manual culture protocol. Three sets of primers and a TaqMan probe were designed to target the tdh, trh, and tlh genes and used for real-time PCR. This study was successful in isolating V. parahaemolyticus from a mussel sample and detecting two of its genes (tdh and tlh) in food and pure culture by real-time PCR.

Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters

“…Pathogenic V. parahaemolyticus bacteria in the environment and food samples typically comprise 0.3 to 3% of the total V. parahaemolyticus population (4,8,20,22,43). DNA-based assays targeting these genes were developed for the detection and enumeration of V. parahaemolyticus bacteria (7,10,21,26,27,30,38,44). However, the ability of the PCR assays to detect low levels of pathogenic V. parahaemolyticus bacteria in the presence of a high background of nonpathogenic V. parahaemolyticus bacteria was not reported by the authors (10,30,44).…”

“…DNA-based assays targeting these genes were developed for the detection and enumeration of V. parahaemolyticus bacteria (7,10,21,26,27,30,38,44). However, the ability of the PCR assays to detect low levels of pathogenic V. parahaemolyticus bacteria in the presence of a high background of nonpathogenic V. parahaemolyticus bacteria was not reported by the authors (10,30,44). The preferential amplification of nonpathogenic members of the total V. parahaemolyticus population due to the muchhigher copy number of the target could preclude the detection of pathogenic strains present in lower numbers.…”

Detection ofVibrio parahaemolyticusin Shellfish by Use of Multiplexed Real-Time PCR with TaqMan Fluorescent Probes

“…Successful real-time PCR for V. parahaemolyticus using a TaqMan probe have targeted a single locus (tdh) (8) or dual loci in a multiplexed format (tdh and toxR) (27). For a recent food-borne outbreak that originated from a Chinese buffet in Polk County, FL, V. parahaemolyticus strains containing tlh and tdh were identified in mussels using real-time PCR with TaqMan fluorescent probes (16). However, in this study the workers tested each targeted gene in separate reactions and did not test for the pandemic strain of the V. parahaemolyticus O3:K6 serotype.…”

Vibrio spp. isolados de mamíferos marinhos capturados na região litorânea do Sudeste ao Sul do Brasil

“…A redução da oferta de alimentos força o deslocamento dos mamíferos marinhos para zonas muito afastadas de seu nicho ecológico natural, favorecendo a pesca predatória indiscriminada, a captura acidental em redes de pesca ou situações de encalhe em bancos de areia. Este aspecto, associado à poluição ambiental e outras modificações antropogênicas, contribui para o aumento da possibilidade de veiculação de patógenos de habitat aquático (Cavalcanti 2003, Davis et al 2004, MMWR, 2005.…”