基于内标准基因的云南野生菌等温检测新技术研究

野生菌种属鉴定及快速检测，是开发种质资源、防止毒蕈中毒的关键技术。云南野生菌资源丰富，食入未知野生菌而导致中毒时有发生，由于形态学鉴定易受外界因素影响、难区分形态差异小的菌株以及rDNA-ITS方法的成功率取决于阳性样品种类、无法区分混合物等缺点，基于内标准基因在生物源成分鉴定的优势，筛选野生菌内标准基因以进行种属鉴定。同时，对同种野生菌同段DNA序列进行SNP筛选，以实现野生菌种内鉴定。等温扩增是应用较广的快检手段，申请人开发的SUP-MLPA可实现高通量特异性检测，特别是SNP检测。但MLPA为变温扩增，为满足等温过程，本研究改进其反应体系。根据内标准基因和SNP位点设计引物和探针，优化It-MLPA反应条件，建立野生菌定性和Taqman探针定量等温快速检测体系。该研究一方面提高野生菌种属鉴定准确性，为其中毒的临床诊断提供依据；也为野生菌乃至更多食品生物源掺假提供精准快速的检测方法。

The species-specific identification and rapid detection of the wild mushrooms are the key techniques for development of germplasm resources and prevention of toadstool poisoning. Yunnan Province is a region rich in wild mushrooms, and the poison incident caused by the ingestion of unknown wild fungus usually happens. There are lots of disadvantages in the existed mushroom identification methods, including morphological identification and rDNA-ITS analysis, such as vulnerable to the external factors, difficult to distinguish the strains with little differences and the mixed samples, and the rate of the success is mainly depended on the types of positive samples. Based on the advantage of endogenous reference gene in the biological component identification, the species-specific endogenous reference genes of the wild mushrooms are screened. At the same time, the SNP sites on the same DNA sequence within the same species of wild mushroom are selected to realize the intraspecific identification. The isothermal amplification is widely used in the on-site rapid detection, the SUP-MLPA developed by the applicant have a high throughput and excellent specificity, especially for detecting SNP site. However, MLPA is not an isothermal amplification, in order to satisfy the rapid detection, the reaction system is improved. The primers and probes are designed based on the sequences of the endogenous reference genes and SNPs, the isothermal rapid qualitative and quantitative detection technique with the help of Taqman probe is established through optimizing the conditions. While this study can effectively enhance the accuracy of the species-specific identifications of the wild mushrooms and provide the technical support for the clinical diagnosis of the toadstool poisoning; on the other hand, the established quantitative It-MLPA detection can provide a specific, time- and cost- saving method for the biological adulteration in wild mushrooms, and even in more agricultural products.