Quantification of Vibrio vulnificus in shellfish by conventional *PCR and real time PCR

• Main Author: Wang, Shishan
• Language: ENG
• Published: ScholarWorks@UMass Amherst 2006
• Subjects: Food science|Microbiology
• Online Access: https://scholarworks.umass.edu/dissertations/AAI3242363

Vibrio vulnificus is a Gram-negative estuarine bacterium that can cause wound infection, primary septicemia and gastroenteritis in susceptible individuals. Since it is a notably lethal pathogen associated with seafood, rapid detection and quantification of V. vulnificus is an important issue for the protection of consumer health and the seafood industry. With conventional PCR the minimum detection level with a new nucleic acid dye (GelStar stain) was 16 CFU per PCR reaction for pure culture compared to 40 CFU with ethidium bromide (EB). With real time PCR, the detection limit was 100 CFU/g of tissue with a linear detection range of 102 to 108 CFU/g without enrichment and 1 CFU with a detection range of 1 to 106 CFU/g of tissue after 5h enrichment. A 508-bp DNA fragment was used as an internal standard for competition with target DNA in competitive PCR, by which the detection sensitivity was 220 CFU of pure culture per PCR reaction, 2700 CFU/PCR with shellfish tissue without enrichment, and 7 CFU/PCR with shellfish tissue after 10 h enrichment. Geobacillus stearothermophilus and hemoglobin were used as a reference strain and a PCR inhibitor respectively, to establish comparative real time PCR, by which the minimum detection sensitivity was 102 CFU per ml of pure culture and 103 CFU per gram of clam tissue. The equation (Ct, T0 = Ct,TX - ΔCt) can be used for enumeration of V. vulnificus in tissue in conjunction with a standard curve relating Ct values to Log genomic targets in the absence of PCR inhibitors via comparative RT-PCR. Amplification of DNA from heat killed cells in a mixture with viable cells was successfully inhibited by treatment of the cells with 2.5 μg/ml of ethidium bromide monoazide (EMA) followed by 15 min photolysis, whereas the DNA from viable cells present was successfully amplified by real time PCR. Using this technique, the detection sensitivity for viable bacterial cells was 10 CFU per ml of pure culture and 100 CFU/g of clam tissue.