Title page for ETD etd-04102007-131052

Type of Document Master's Thesis
Author Senevirathne, Reshani Nisansala
Author's Email Address rsenev1@lsu.edu
URN etd-04102007-131052
Title Detection of Vibrio vulnificus by Direct Colony Immunoblot
Degree Master of Science (M.S.)
Department Food Science
Advisory Committee
Advisor Name Title
Marlene E. Janes Committee Chair
Jon W. Bell Committee Member
Witoon Prinyawiwatkul Committee Member
  • Vibrio vulnificus
  • Oysters
  • Detection
  • Enumeration
  • Direct Colony Immunoblot
  • Colony Immunoblot
  • Immunoblot
Date of Defense 2007-03-26
Availability unrestricted
Vibrio vulnificus (Vv) is a natural occurring bacterium of world wide estuarine environments, which concentrate on filter feeding shellfish. Under cooked seafood contaminated with this pathogen is a leading cause of 95% of seafood-related, foodborne deaths. The development of a rapid, reliable and user-friendly method for Vv enumeration would help to reduce mortality rate. A direct colony immunoblot (DCI) method was developed and used as a rapid enumeration with high sensitivity and the specificity. This method was optimized using Vv agar plates incubated for 16 h at 35 °C. Colonies were transferred from incubated plates to polyvinylidene fluoride (PVDF) membranes and treated with rabbit anti–flagellar Vv antibodies for 1 h, then washed 3 times. The membranes were then incubated with peroxidase-conjugated goat anti-rabbit IgG for 1 h, and washed 3 times. Finally, the color development mixture was added (Tris buffer, 3, 3’-diaminobenzidine, NiCl2 and H2O2) for 5 min. Positive colonies produced a purple color. Total time duration for enumeration of Vv by the DCI was 3.5 h. The DCI method was compared with the FDA recommended DNA probe hybridization (DNAH) method (6-10 h) and most probable number MPN method (50 h) for enumeration of naturally occurring Vv in oysters. There was no significant difference between the DCI and DNAH methods at 0h, 4h, 8h, 12h and 24h, with both methods having Vv counts of about 2.90 Log CFU/g. By day 7 there was a significance difference between the two methods, with the DNAH exhibiting higher Vv counts (2.62 Log CFU/g) compared to the DCI (2.22 Log CFU/g). By 14 days the counts for both methods were not significantly different from each other (1 Log CFU/g). The DCI method exhibited comparable Vv counts in raw oysters compared with those of the DNAH method except for day 7, which may be due to false positive colonies detected by the DNAH method. The DCI could be a more reliable, inexpensive, rapid and user-friendly method for enumeration of Vv in raw oysters. This could possibly be used as a rapid enumeration method by regulatory agencies or the seafood industry.

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