Abstract

In order to achieve rapid detection of microbial pathogens, such as V. vulnificus and V. parahaemolyticus, it is necessary to develop, test, and implement technologically advanced, rapid, robust, sensitive, and quantitative methods that are in compliance with the recent ISSC guidelines. In this proposed research, we plan to develop and validate state-of-the-art methodologies, such as phage-displayed species-specific ligands/Fluorescent Activated Cell Sorter, and MPN/Real-time PCR for rapid, reliable, and quantitative methods of detection of these pathogens in PHT shellfish. The proposed research will compare these detection methodologies for their applicability and effectiveness towards the detection of these pathogens in PHT oysters.

The first objective is to identify and characterize phage displayed species-specific peptide ligands to establish a rapid and quantitative “whole cell” detection of V. vulnificus and V. parahaemolyticus in shellfish using a fluorescent activated cell sorter (FACS). An Escherichia coli phage M13 with randomly displayed short surface peptides will be used to select V. vulnificus and V. parahaemolyticus-specific ligands using a procedure called Biopanning method. The nucleotide sequence of the short peptide will be determined and an antibody for the peptide will be developed. The antibody will then be labeled with a fluorescent dye. The antibody will be used to “capture” targeted V. vulnificus and V. parahaemolyticus cells from oyster tissue homogenates and a quantitative estimation will be determined using a BD fluorescent activated cell sorter (BD FACS Calibur). The methodology will be tested on post-harvest treated (PHT) oysters. Our preliminary data shows that a peptide ligand that specifically binds to V. vulnificus-specific surface protein can be used for the detection of this pathogen. This method is feasible and has potential to develop a µSERS-based rapid detection of this pathogen without purification of cellular protein or nucleic acids.

The second objective is to develop and validate an MPN/Real-time PCR-based quantitative detection of V. vulnificus and V. parahaemolyticus in shellfish and compare this method with the phage-displayed species-specific peptide ligand/FACS procedure. In this objective, the currently recommended conventional quantitative MPN method of detection will be combined with the real-time PCR to establish a rapid “one-day” assay for a reliable and quantitative detection of these pathogens. Also, comparison of phage-displayed peptide/FACS method with the MPN/real time PCR will help establish an alternative state-of-the-art detection method for these pathogens in shellfish. We have already selected and tested some of the primers and probes necessary to develop this detection method. In a preliminary study, detection of V. vulnificus in shellfish using SYBR Green 1-based real-time PCR could be completed in less than 8 h following 5 h enrichment of the pathogen. This real-time PCR detection will be optimized and tested on a MPN/real-time PCR platform. We expect this approach will be quantitative and in compliance with the current ISSC guidelines. All detection methods will be tested on PHT oysters following the ISSC/FDA standard methods.

These methodologies will be communicated with government and private industry laboratories for further testing and validation study. Rapid and early detection of these pathogens, complying with the required quantitative detection guidelines suggested by the ISSC, will help the seafood industry market “microbiologically safe” oysters for consumption. This will improve consumer confidence in Gulf of Mexico oysters and help the seafood industry and states along the Gulf of Mexico gain higher revenues.

Objectives

The objective is to develop and validate an MPN/Real-time PCR-based quantitative detection of V. vulnificus and V. parahaemolyticus in shellfish and compare this method with the phagedisplayed species-specific peptide ligand/FACS procedure. In this objective, the currently recommended conventional quantitative MPN method of detection will be combined with the real-time PCR to establish a rapid “one-day” assay for a reliable and quantitative detection of these pathogens. Also, comparison of phage-displayed peptide/FACS method with the MPN/real time PCR will help establish an alternative state-of-the-art detection method for these pathogens in shellfish.

Methodology

Appropriate primers and probes for the development of a fluorogenic SYBR Green I or TaqMan®  assay are listed in the text. The reaction parameters are currently being optimized. Detection of V. vulnificus in shellfish using the real-time PCR was possible in less than 8 h following 5 h enrichment. This real-time PCR detection will be optimized and tested on a MPN/real-time PCR platform to establish a rapid and quantitative detection of V. vulnificus and V. parahaemolyticus in shellfish that is in compliance with the current ISSC guidelines. The effectiveness of the methodologies will be tested on PHT oysters.

Rationale

This approach will help establish a quantitative detection of V. vulnificus (less than 3 cells per gram of oyster tissue) and V. arahaemolyticus (less than 10 cells per gram of oyster tissue) in shellfish. The detection will be achieved in real-time and reportable data will be generated within 8 h. We anticipate this approach will be rapid and sensitive for the detection of these pathogens in shellfish. The objective is to develop and validate a rapid and quantitative detection that is in compliance with the recent ISSC guidelines. Rapid detection will help improve the microbiological safety of shellfish, especially the Gulf Coast oysters, which will be beneficial for the shellfish industry in states along the Gulf of Mexico.