Kim Mi-Gyeong, Hong Min Ji, Seo Doo Won, Jung Hyun Mi, Han Hyun-Ja, Kim Seung Hwan, Joo Insun
Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, Ministry of Food and Drug Safety, Cheongju-si, Chungcheongbuk-do, Republic of Korea.
National Institute of Fisheries Science, Incheon, Republic of Korea.
PLoS One. 2025 Apr 25;20(4):e0320724. doi: 10.1371/journal.pone.0320724. eCollection 2025.
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 Cq 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.
异尖线虫病是一种食源性寄生虫感染，由食用含有异尖线虫科第三期幼虫的生的或未煮熟的海鲜引起。这种感染在全球范围内都有发现，在韩国和日本的患病率尤其高。因此，有必要比较和分析最佳检测方法，以预防异尖线虫病的爆发。在本研究中，开发了一种基于Taqman的物种特异性qPCR方法，用于检测简单异尖线虫和欺骗伪新线虫的内转录间隔区和线粒体DNA基因。根据国内外检测方法的数据选择寄生虫特异性引物/探针组。此外，我们还根据每种寄生虫的目标区域设计了自己的引物/探针组。通过全面的文献综述和自主创建过程，为简单异尖线虫和欺骗伪新线虫选择了13套检测方法。然后通过比较提取DNA的Cq值来评估这些方法的灵敏度。然后比较筛选过程中检测到的6套引物/探针的浓度，以优化检测方法。最终优化后的方法显示，简单异尖线虫的检测限为0.0019 ng/µL，欺骗伪新线虫的检测限为0.0001 ng/µL。特异性试验还证实，该方法与5种寄生虫样本和3种异尖线虫质粒DNA没有交叉反应。本研究将有助于异尖线虫病快速检测方法的开发，为积极应对食物中毒爆发提供基础。
