燕窝显微特征及基于DNA条形码的基源鉴别

Microscopic characteristics of edible bird’s nest and identification of its basic source based on DNA barcode

目的建立基于Cytb等5种基因序列的燕窝(edible bird’s nest,EBN)基源DNA条形码鉴别方法。方法以泰国、越南、马来西亚、印度尼西亚等地的20个正品燕窝和1个伪品燕窝为研究对象,利用显微技术对21个样品进行观察,再提取基因组DNA,分别利用5对引物扩增Cytb、ND2、12S、COI和Fib7基因序列并双向测序。聚合酶链式反应(polymerase chain reaction,PCR)产物测序分析后,NCBI中进行BLAST搜索,分析样品遗传距离,利用最大似然法(maximum likelihood,ML)进行系统发育分析。结果正品燕窝和伪品燕窝在形态上具有较明显区别。BLAST分析表明,所获得的Cytb、ND2、12S、COI基因序列均与雨燕科的爪哇金丝燕(Aerodramus fuciphagus)具有最高的序列一致性。系统发育分析表明不同种类的燕窝处于不同分支中,所测定的4个基因序列均与爪哇金丝燕处于同一分支中,显示最近的亲缘关系。结论该方法可用于燕窝基源的分子鉴定,也为燕窝的真伪鉴别及溯源提供理论依据。

Objective To establish the identification method of edible bird’s nest(EBN)based on DNA barcode with 5 kinds of gene sequences such as Cytb.Methods Twenty authentic EBNs samples and a fake EBNs sample from Thailand,Vietnam,Malaysia and Indonesia were selected as the research subjects,twenty-one samples were observed by microscopy,and genomic DNA were extracted,Cytb,ND2,12S,COI and Fib7 gene sequences were amplified and bidirectionally sequenced by 5 pairs of primers,respectively.After sequencing and analysis of polymerase chain reaction(PCR)products,they were input to NCBI for BLAST search,where the genetic distance of samples were analyzed,and phylogenetic analysis were conducted using maximum likelihood method(ML).Results There were significant differences in morphology between authentic and fake EBN.BLAST analysis showed that the sequences of Cytb,ND2,12S and COI genes obtained had the highest sequence consistency with Aerodramus fuciphagus in swifts family.Phylogenetic analysis revealed that different species of EBN were in different branches,and the 4 gene sequences determined were all in the same branch as Aerodramus fuciphagus,showing the closest genetic relationship.Conclusion This method can be used for molecular identification of EBN samples,and also provide theoretical basis for original identification and traceability of EBN.