rDNA-ITS分子条形码鉴定技术用于亚稀褶黑菇鉴定的研究

Study on identification of Russula subnigricans by rDNA-ITS molecular barcode technique

目的建立一种可靠的内转录间隔区(ITS)分子条形码标记技术,作为亚稀褶黑菇(Russula subnigricans)中毒鉴定的有效方法。方法子实体中DNA被提取后,经过PCR扩增、克隆后再测序,得到ITS全序列;将DNA序列与GenBank中序列进行BLAST比较,获得二者间的同源性,建立系统发育树并进行分析。对模板DNA浓度、取样部位、烹调的影响等条件进行评价。结果在3个野生蘑菇样品中,引起中毒的蘑菇样品(R02)与GenBank中7个亚稀褶黑菇亚种的ITS序列同源性达99%,且系统发育树显示与这些亚种处在同一支,鉴定为亚稀褶黑菇,与形态鉴定结果一致;另外两个样品不属于亚稀褶黑菇。结论建立了rDNA-ITS分子条形码鉴定方法,可用于亚稀褶黑菇的准确鉴定,将为毒蘑菇中毒事件的应急处置提供科学的技术手段。

Objective To develop a reliable ITS(internal transcribed spacer)sequencing method to identify food poisoning incident caused by toxic mushroom(Russula subnigricans).Methods DNA extracted from the mushroom powder was amplified by PCR technique,followed by ITS sequencing through cloning technology.The similarity was compared between the DNA sequences of the sample and from GenBank in NCBI database with BLAST(Basic Local Alignment Search Tool)and the phylogenetic tree was built for further analysis.Some factors such as DNA concentration,sampling site and cooking influence were also evaluated.Results The similarity was more than 99%between the DNA sequences of the poisoning sample(R02)and the DNA sequences of seven kinds of toxic mushroom(Russula subnigricans)subgroups from GenBank,and all the DNA sequences belonged to the same branch,which showed that the poisoning sample and the corresponding species belonged to the same species.Coupled with morphological identification,the sample R02 was identified as Russula subnigricans.The other two samples did not belong to Russula subnigricans.Conclusion The results showed that the rDNA-ITS molecular barcode technique was successfully developed to identify the toxic mushroom,such as Russula subnigricans,and will be an effective identification method for food poisonings caused by mushroom.