摘要
【目的】构建简单异尖线虫(Anisakis simplex)、费氏宫脂线虫(Hysterothylacium fabri)、内弯宫脂线虫(Hysterothylacium aduncum)、拟地新线虫(Pseudoterranova decipiens)、典型异尖线虫(Anisakis typica)、带鱼针蛔线虫(Raphidascaris trichiuri)、厦门宫脂线虫(Hysterothylacium amoyense)和中华宫脂线虫(Hysterothylacium sinense)的快速检测方法,实现对以上8种异尖线虫的高通量检测及准确分型。【方法】采集8种异尖线虫核酸进行凝胶电泳,测序并进行序列对比,设计引物和TaqMan荧光探针;通过优化体系及退火温度,建立多重实时荧光PCR检测体系,并添加海产品核酸背景验证其特异性和灵敏度。【结果】建立了基于多重实时荧光PCR技术针对海产品中异尖线虫的快速检测鉴定方法,可在一个反应体系里同时检测鉴定4种异尖线虫,双通道反应体系可一次性检测鉴定8种异尖线虫。体系中,引物和探针的终浓度分别为0.60、0.20μmol/L,DNA灵敏度为103拷贝/mL;对带鱼(Trichiurus lepturus)、鳕鱼(Gadus)、舟山宫脂线虫(Hysterothylacium zhoushanensis)、棘颚口线虫(Gnathostoma spinigerum)均无荧光信号;针对等量混合的4种异尖线虫模拟样本均可检出,检出限为1.0%。【结论】建立的海产品异尖线虫多重实时荧光PCR快速检测方法操作简便、灵敏度高、特异性强、重复性好,可应用于日常食品检验和异尖线虫分型鉴别研究。
【Objective】To establish a method for the high-throughput detection,and identification of Anisakis simplex,Hysterothylacium fabri,Hysterothylacium aduncum,Pseudoterranova decipiens,Anisakis typica,Raphidascaris trichiurid,Hysterothylacium amoyense,and Hysterothylacium sinense.【Methods】The nucleic acid of 8 species of anisakid nematodes was collected and detected by gel electrophoresis and sent for sequenced comparison.Using the conservation and specificity of ITS(Internal transcribed spacer)sequences,primers and TaqMan fluorescent probes were designed.By optimizing the system and annealing temperature,a multiplex real-time fluorescence PCR detection system was established,and the seafood nucleic acid background was added to verify its specificity,sensitivity,and other related technical parameters.【Results】A rapid detection and identification technology for anisakid nematodes in seafood was established based on multiplex real-time fluorescence PCR technology,which could simultaneously detect 4 species of anisakid nematodes in one reaction system.The dual-channel multiplex real-time PCR reaction system can simultaneously detect and identify the 8 species of anisakid nematodes in one experiment.In the system,the final concentrations of primers and probes were 0.60 and 0.20μmol/L,respectively;The sensitiviyt of multiplex Real-time fluorescence PCR was 103 copies/mL;Except for the worm species,there was no fluorescent signal for Trichiurus lepturus,Gadus,Hysterothylacium zhoushanensis,and Gnathostoma spinigerum;The simulated samples of 4 species of anisakid nematodes could be detected with the detection limit of 1.0%.【Conclusion】The established method based on multiplex real-time fluorescence PCR technology for the rapid detection of anisakid nematodes in seafood is easy to operate,with high sensitivity,strong specificity and good repeatability,and can be used in daily food inspection and anisakid nematodes classification research.
作者
黄姝玲
HUANG Shu-ling(Fujian Inspection and Research Institute for Product Quality,Fuzhou 350002,China)
出处
《广东海洋大学学报》
CAS
北大核心
2022年第6期122-129,共8页
Journal of Guangdong Ocean University
基金
国家市场监督管理总局科技计划项目(No.2019MK037)。
关键词
异尖线虫
海产品
快速检测鉴定
多重实时荧光PCR
Anisakid nematodes
seafood
rapid detection and identification
multiplex real-time fluorescence PCR
