黑松露成分TaqMan探针实时荧光PCR检测方法的建立与应用
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(1.大连海关技术中心 辽宁大连 116001 ;2.中华全国供销合作总社昆明食用菌研究所 昆明 650033)

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海关技术规范制(修)订计划项目(2020B160);海关总署科研项目(2021HK189);大连海关科研项目(2021DK10,2022DK05)


Development and Application of a TaqMan Real-time PCR Assay for Rapid Detection of Black Truffles Derived Ingredients
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(1.Technology Center of Dalian Customs District, Dalian 116001, Liaoning;2.Kunming Edible Fungi Institute of All China Federation of Supply and Marketing Cooperatives, Kunming 650033)

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    摘要:

    以黑松露核糖体内转录间隔区基因为靶基因设计特异性引物和探针,建立黑松露成分的TaqMan实时荧光PCR检测方法,并进行物种特异性验证。同时对不同掺入比例的模拟样品和市售黑松露及制品进行检测。结果表明:该方法特异性强,仅对黑松露基因组DNA出现特异性扩增曲线,与其它食用菌和异源性动植物样品无非目标扩增;检测灵敏度为1×10-3 ng/μL黑松露基因组DNA或质量分数0.01%的黑松露粉。对120份市售黑松露(鲜品、冻品、干品、粉末)、黑松露制品(黑松露酱和黑松露调味粉)以及不含黑松露成分的杂菌包进行检测,在70份黑松露(鲜品、冻品、干品、粉末)和10份黑松露制品中检出黑松露成分,其它样品均未检出黑松露成分,检测结果与商品标识一致。该方法灵敏度高,特异性强,可快速精准地进行黑松露成分的真实性甄别。

    Abstract:

    In this study, a TaqMan real-time PCR method was established for rapid detection of black truffle derived ingredients. Specific primers and probes were designed according to conserved sequence of ITS gene of Tuber indicum complex to test the target gene fragment in the samples, and species-specific detection, sensitivity detection and practical application detection were carried out. Results showed that this real-time PCR method had strong specificity, only showed specific amplification curves for black truffle genomic DNA, but there was no amplification curve for other edible fungi, animal and plant materials. The limit of detection was 1.0×10-3 ng/μL genomic DNA or 0.01% (mass fraction) per reaction. The feasibility of the method was further verified by detecting 120 commercial black truffles (fresh, frozen, dry and powder), black truffle products (black truffle sauce and black truffle seasoning powder) and miscellaneous bacteria bags without black truffle components. The black truffle components were detected in 70 black truffles and 10 black truffle products. This method has the characteristics of strong specificity, high sensitivity, rapidity and high sufficiency, and is suitable for rapid identification of black truffles ingredients.

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杨爱馥,孙赟,万超,刘雪华,邰丽梅,王玫.黑松露成分TaqMan探针实时荧光PCR检测方法的建立与应用[J].中国食品学报,2023,23(7):403-409

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  • 收稿日期:2022-07-27
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  • 在线发布日期: 2023-08-17
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