Objectives

The objective of the proposal is to develop an easy-to-use nucleic acid-based molecular diagnostic assay for the detection of Vibrio vulnificus in post-harvest processed (PHP) oysters using a thermostable helicase-mediated isothermal DNA amplification (HDA) and an Isoamp™ disposable on Demand Lateral Flow device technology. The proposed assay does not require an expensive real-time PCR thermal cycler, therefore it is cost-effective and could easily be adopted by the seafood industry or could potentially be used on field samples.

Methodology

The isothermal HDA technology (BioHelix, Corp.) uses an enzyme called thermostable DNA helicase (tHDA) to separate DNA strands during in vitro synthesis of nucleic acid at an isothermal condition (~65°C constant). Unlike other isothermal reactions, Biohelix’s HDA duplicates the targeted gene and a Competitive Internal Control (CIC) DNA (BioHelix) as seen in living cells. HDA has the advantage over PCR thermal cycling as it uses simple enzymology for the amplification of the targeted gene, an IC DNA in an isothermal condition. The amplicons are then detected in a disposable on Demand Lateral Flow device, which is designed to analyze a single amplification reaction containing up to two different amplicons (V. vulnificus vvhA and CIC DNA). 

The strips in the device are coated with detection reagents consisting of latex beads conjugated to streptavidin. CIC amplicons will be labeled with a 5’-biotinylated (BIO) amplification primer, and hybridized to a 3’ dioxygenin labeled probe, while vvhA amplicons will be labeled with the same 5’BIO primer and hybridized to a 3’ FITC labeled probe. The PHP oysters will be obtained from Ameripure Processing Company, Franklin, LA. Ameripure’s raw oysters are harvested from “approved” waters of the Gulf of Mexico, meeting the strict requirements of the NSSP, and undergo post harvest processing by a patented method of controlled heat and cold temperature treatments to reduce Vibrio bacterial load to undetectable levels. These in-shell raw PHP oysters will be subjected to 3-tube MPN enrichment. The MPN positive tubes will be tested for V. vulnificus levels using the HDA/on-Demand Lateral Flow assay. BioHelix and AmeriPure have agreed to partner with this project. 

Rationale

In the U.S., shellfish harvest supports a significant commercial industry with ~27.0 million-pounds of meat with an approximate market value of $72.7 million. However, warm waters in the Gulf of Mexico harbors pathogenic bacteria such as V. vulnificus. Infection by V. vulnificus due to the consumption of raw, unprocessed oysters often results in fatal consequences to individuals with underlying liver disease or those who are otherwise immunocompromised. The oyster industry in the U.S., in general, is currently the target of consumer groups concerned with the risk of illnesses associated with raw oyster consumption. In order to establish steady consumer confidence, which is the foundation for a stable economy for the shellfish industry, the ISSC has mandated measures for public education along with the development, validation and implementation of methodologies for reduction, and rapid detection of harmful microbial pathogens in oysters through technological innovations and adoptions. The proposal presents a cost-effective and simple-to-use HDA/IsoAmp on Demand Lateral Flow detection assay to monitor reduction of V. vulnificus in PHP oysters to non-detectable levels. This assay will help producers reduce overhead costs; maintain steady supply safe-to-eat raw oysters and improve the industry’s public image as well as the safety of the consumers of raw oysters.