Development and Evaluation of an Immunochromatographic Rapid Assay for the Detection of Pathogenic Vibrio parahaemolyticus in Food

Introduction: Vibrio parahaemolyticusis a Gram-negative, halophilic bacterium of the estuarine environment and a major cause of foodborne illness from consumption of raw or undercooked seafood, particularly in Asia. Standard detection methods are mainly cultural based, labor-intensive and time-consuming. Rapid deterioration of raw seafood in particular, requires faster detection methods. Lateral Flow technology offers a reliable, fast, user-friendly, alternative detection method.

Purpose: To develop and evaluate an immunochromatographic assay for detection of pathogenic Vibrio parahaemolyticus from food, as a rapid alternative to standard reference methods.

Methods: A sandwich Lateral Flow assay was developed, using gold-labeled specific antibodies for detection of thermostable direct hemolysin (TDH) of Vibrio parahaemolyticus. Evaluation was by determination of pure culture detection limit, sensitivity and specificity in fresh and frozen seafood samples artificially contaminated with V. parahaemolyticus at 10¹ to 10⁴ CFU/g, inclusivity testing of tdh-positive V. parahaemolyticus strains and exclusivity testing of tdh-negative V. parahaemolyticusstrains, other Vibrios and non-Vibrios. Reference method comparison was ISO/TS 21872-1:2007.

Results: Pure culture detection limit of 4 strains of V. parahaemolyticus was 125 pg/ml. 81% Inclusivity demonstrated from testing 23 tdh-positive V. parahaemolyticus strains. 100% Exclusivity demonstrated from testing 69 tdh-negative V. parahaemolyticus strains, other Vibrios and non-Vibrios. V. parahaemolyticus was detected in fresh food samples artificially contaminated with 10¹ to 10² CFU/g and in frozen samples at 10³ to 10⁴ CFU/g. After 24 h sample enrichment and implementation of a pre-treatment step (centrifugation), sensitivity and specificity of 100% was attained. Performance was equivalent to the culture-based ISO reference method. The LFA reduced time-to-result to a 24 h enrichment plus 1 h sample pre-treatment and assay performance.

Significance: The developed Lateral Flow Assay provides a unique, alternative, fast and simple method for detection of pathogenic V. parahaemolyticus from food.