Comparative study of Taqman-based qPCR assay for the detection of Anisakis simplex and Pseudoterranova decipiens

Abstract

Anisakidosis is a foodborne parasitic infection caused by the consumption of raw or uncooked seafood that contains third stage larvae from the Anisakidae family. This infection has been observed across the globe, with a particularly high prevalence in South Korea and Japan. Consequently, there is a necessity to compare and analyze the optimal detection methods with a view to preventing Anisakis outbreaks. In this study, a species-specific Taqman-based qPCR method was developed for the detection of the internal transcribed spacer region and mtDNA genes of Anisakis simplex and Pseudoterranova decipiens. Parasite-specific primer/probe sets were selected based on the data from domestic and foreign detection methods. In addition, we have designed our own primer/probe sets based on the target region of each parasite. A comprehensive literature review and a self-creation process were undertaken to select thirteen detection method sets for A. simplex and P. decipiens. The sensitivity of these sets was then evaluated by comparing the C_q values from extracted DNA. The concentrations of six primer/probe sets detected through the screening process were then compared to optimize the test method. The resultant optimized method demonstrated a limit of detection of 0.0019 ng/µL for A. simplex and 0.0001 ng/µL for P. decipiens. The specificity test also confirmed that there was no cross-activity with the five parasite samples and the three types of anisakids plasmid DNA. This study would contribute development of a rapid detection method for anisakidosis, providing a foundation for proactive responses to food poisoning outbreaks.