目的 以河北省常见鼠疫媒介蚤为研究对象，使用线粒体细胞色素C氧化酶亚基Ⅰ（COⅠ）基因和细胞色素C氧化酶亚基Ⅱ（COⅡ）基因对其进行分子生物学鉴定，建立河北省常见蚤种的DNA条形码数据库。方法 2018年6－8月在张家口市康保牧场采集鼠体寄生蚤及洞干蚤，2018年9－10月在张家口崇礼区采集鼠体寄生蚤，经形态学鉴定后，选取标本形态完整的不同蚤种提取总DNA，扩增线粒体COⅠ和COⅡ基因片段，并进行测序，将测序结果与GenBank中蚤类序列进行BLAST同源性比对，利用邻接法构建COⅠ和COⅡ基因序列的系统进化树。结果 选取的16份蚤类样本均能通过PCR扩增出特异性COⅠ和COⅡ基因条带，其中13份样本COⅠ测序成功，12份样本COⅡ测序成功。COⅠ基因的分子进化树结果中，12份样本与形态学鉴定结果一致，1份样本与形态学有差异。COⅡ基因的分子进化树结果与形态学鉴定全部一致。结论 COⅠ和COⅡ基因均可作为DNA条形编码基因对河北省鼠疫疫源地的常见蚤类进行物种鉴定，利用2个基因同时进行蚤种鉴定提高了鉴定的成功率，所积累数据为以后的蚤类分子鉴定技术提供了可利用的数据库。
兰晓宇, 鲁亮, 候芝林, 杜国义, 史献明, 崔耀仁, 刘冠纯, 陈永明, 康东梅, 郑楠, 任兴宇, 闫东
. DNA条形码技术在河北省鼠疫疫源地常见蚤种鉴定中的应用[J]. 中国媒介生物学及控制杂志, 2020
, 31(6)
: 662
-666
.
DOI: 10.11853/j.issn.1003.8280.2020.06.007
Objective To perform molecular biological identification of the common plague vector fleas in Hebei province, China by analyzing the mitochondrial cytochrome c oxidase subunitⅠ(COⅠ) gene and cytochrome c oxidase subunit Ⅱ:(COⅡ) gene, and to establish a DNA barcode database of common flea species in Hebei province. Methods Rodent-parasitic fleas and burrow fleas were collected in Kangbao ranch of Zhangjiakou, Hebei province from June to August, 2018, and rodent-parasitic fleas were collected in Chongli district of Zhangjiakou from September to October, 2018. After morphological identification, total DNA was extracted from various species of fleas of intact specimens, and then the mitochondrial COⅠ and COⅡ gene fragments were amplified and sequenced. The sequences were compared by BLAST for homology with those of fleas in GenBank. The phylogenetic tree of the COⅠ gene and COⅡ gene sequences was constructed by the neighbor-joining method. Results The specific COⅠ and COⅡ gene bands were amplified by PCR in all 16 samples of fleas. Among them, the COⅠ gene was sequenced successfully in 13 samples, and the COⅡ gene in 12 samples. According to the results of molecular phylogenetic tree of the COⅠ gene, 12 samples were consistent with morphological identification, and 1 sample was not. The results of molecular phylogenetic tree of the COⅡ gene were all consistent with the morphological identification in 12 samples. Conclusion Both COⅠ and COⅡ genes can be used as DNA barcode markers for species identification of common fleas in plague foci of Hebei province. Simultaneous identification of fleas using two genes improves the success rate of identification. The accumulated data will help establish an available database for future flea molecular identification technology.
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