实时荧光聚合酶链式反应法鉴定螺蛳粉螺蛳肉汤包中圆田螺和环棱螺

Identification of Cipangopaludina and Bellamya in the meat soup package of river snail rice noodle by real-time fluorescence polymerase chain reaction

中文关键词:  圆田螺属  环棱螺属  螺蛳粉  实时荧光聚合酶链式反应法

英文关键词:Cipangopaludina  Bellamya  river snail rice noodle  real-time fluorescence polymerase chain reaction

中文摘要:

      目的 基于实时荧光聚合酶链式反应(polymerase chain reaction, PCR)法建立一套鉴定圆田螺和环棱螺的方法, 并对螺蛳粉中螺蛳肉汤包的螺源性成分进行鉴定。方法 分别以圆田螺属和环棱螺属的线粒体基因, 设计特异性引物和探针, 从方法的特异性、灵敏性和重复性考察建立的实时荧光PCR法的有效性。结果 本研究所建立的实时荧光PCR鉴定方法特异性强, 仅对圆田螺属和环棱螺属的DNA在荧光通道有荧光信号检出, 且出现典型扩增曲线, 对多种水产品、家禽家畜、香料调味料原料等异源性动植物基因组DNA在荧光通道均无荧光信号检出, 也无明显的扩增曲线; 该方法对圆田螺属的检测灵敏度低至0.002 ng/μL, 对环棱螺属的检测灵敏度低至0.001 ng/μL; 应用该方法对商场超市流通领域中的21批次不同品牌预包装螺蛳粉中的螺蛳肉汤包提取DNA进行检测。其中7批次不同品牌预包装螺蛳粉中螺蛳肉汤包的DNA检出圆田螺属成分, 占比为33.3%; 18批次不同品牌预包装螺蛳粉中螺蛳肉汤包的DNA检出环棱螺属成分, 占比为85.7%; 3批次不同品牌预包装螺蛳粉中螺蛳肉汤包的DNA均未检出圆田螺属成分和环棱螺属成分, 占比为14.3%; 7批次不同品牌预包装螺蛳粉中螺蛳肉汤包的DNA均检出圆田螺属成分和环棱螺属成分, 占比为33.3%。结论 该方法特异性强, 灵敏度高, 可有效对螺蛳粉中螺蛳肉汤包的螺源性成分进行鉴定, 为市场监管提供有力的技术支持。

英文摘要:

      Objective To establish a method based on real-time fluorescent polymerase chain reaction (PCR) for the identification of the genus Cipangopaludina and Bellamya, and identify the snail-derived ingredients in the meat soup package of river snail rice noodle. Methods Specific primers and probes were designed using mitochondrial genes from the genera Cipangopaludina and Bellamya, respectively. The effectiveness of the established real-time fluorescence PCR method was evaluated for its specificity, sensitivity and repeatability. Results The real-time fluorescence PCR identification method developed in our study exhibited strong specificity, detecting fluorescence signals only in the designated channel for the DNA of the Cipangopaludina and Bellamya, with typical amplification curves observed. No fluorescence signals were detected in the designated channel for the genomic DNA of various heterogeneous animals and plants, including aquatic products, poultry, livestock, and spice and seasoning raw materials, with no observable amplification curves. The method demonstrated sensitivity as low as 0.002 ng/μL for Cipangopaludina and 0.001 ng/μL for Bellamya. The method was employed to analyze DNA extracted from snail meat soup packets in 21 batches of prepackaged river snail rice noodle across various brands in supermarket distribution. In 7 batches of prepackaged river snail rice noodle from different brands, Cipangopaludina components were detected in the DNA of the snail meat soup packets, representing 33.3%. In 18 batches of prepackaged river snail rice noodle from different brands, Bellamya components were detected in the DNA of the snail meat soup packets, representing 85.7%. In 3 batches of prepackaged river snail rice noodle from different brands, neither Cipangopaludina and Bellamya components were detected in the DNA of the snail meat soup packets, accounting for 14.3%. In 7 batches of prepackaged river snail rice noodle from different brands, both Cipangopaludina and Bellamya components were detected in the DNA of the snail meat soup packets, representing 33.3%. Conclusion This method boasts strong specificity and high sensitivity, effectively identifying snail-derived components in snail meat soup packets of river snail rice noodle, thereby providing robust technical support for market regulation.