荧光PCR方法定量检测食品中香菇成分

Quantitative PCR methods for the detection of Lentinus edodes in food

目的建立定量检测食品中香菇成分的荧光聚合酶链式反应(polymerase chain reaction,PCR)法,以辨别出口商品和国内市场中谎报香菇成分含量、以次充好的菌菇产品。方法选取香菇基因组单拷贝核基因Hydrophobin Protein设计香菇种属特异性引物,扩增107 bp的片段,用于荧光PCR定量检测。分别以3种香菇作为阳性对照和14种非香菇菌种、植物产品作为阴性对照,测试实时荧光PCR引物和探针的特异性。以香菇标准DNA进行8个浓度梯度稀释,测试的方法绝对定量限,并制备12个梯度含量的香菇DNA样品,测试香菇相对含量的标准曲线。结果本研究建立的香菇荧光PCR定量检测方法对香菇物种的特异性好,与其他食品物种无交叉结果,荧光PCR扩增效率为0.90,绝对定量限(limit of quantitation,LOQ)为0.01 pg/μL,含量检测LOQ为0.05%。结论本方法重复性和实用性好,可以满足食品和调味料中香菇含量定量检测要求。

Objective To establish a fluorescence polymerase chain reaction(PCR) method for the quantitative detection of Lentinus edodes in food,so as to identify the falsely reported Lentinus edodes in export commodities and domestic market.Methods A single copy nuclear gene of Hydrophobin Protein from Lentinus edodes genome was used to design a pairs species-specific primers and probe for fluorescent PCR quantitative detection of Lentinus edodes,and a 107 bp fragment was amplified for quantitative detection by fluorescence PCR.Three species of Lentinus edodes samples were used as a positive control and 14 species of non-Lentinus edodes strains and plant products were used as negative controls to test the specificity of the primers and probes.Eight concentration gradient dilutions were carried out by the standard DNA of Lentinus edodes for the absolutely quantified test,and 12 gradients of different Lentinus edodes content samples were prepared for test standard curve of the relative content of the Lentinus edodes.Results This fluorescence PCR quantitative detection method established in this study had good specificity for Lentinus edodes species,and there was no crossover between Lentinus edodes species and other food species.The amplification efficiency of fluorescent PCR was 0.90,and the absolute value limit of quantitation(LOQ)was 0.01 pg/μL and the content detection LOQ was 0.05%.Conclusion This method has good repeatability and operability,which can meet the quantitative detection requirements of Lentinus edodes content in food.