基于COⅠ基因的媒介蝇类快速分子鉴定研究  

Study on rapid molecular identification of vector flies based on COⅠ gene

作　　者：王颖 鲁闽 李金庆 刘明杰 岳巧云 

机构地区：[1]烟台出入境检验检疫局技术中心,山东烟台264000 [2]中山出入境检验检疫局

出　　处：《中国媒介生物学及控制杂志》2014年第5期427-431,共5页Chinese Journal of Vector Biology and Control

基　　金：国家科技支撑计划课题(2012BAK11B05);国家质检总局科研基金(2012IK223);山东出入境检验检疫局科研基金(SK201116)~~

摘　　要：目的为解决蝇类形态学鉴定的不足,建立基于细胞色素C氧化酶亚基Ⅰ(COⅠ)基因的DNA条形码技术对蝇类进行分子鉴定。方法选取烟台口岸常见的16只蝇类作为研究对象,形态学鉴定后,取蝇的单后足代表微量组织,提取总DNA。依据资料,选取COⅠ国际通用引物LCO1490:5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′,HCO2198:5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′,建立PCR体系,完善PCR程序,有效扩增蝇类的COⅠ基因。纯化PCR产物后进行序列测定和比对。此外,应用Mega5.05软件分别用最大似然法、邻接法、最大简约法分析系统发育,构建系统发育树比较进化关系。结果 16只蝇类COⅠ基因扩增片段长度为690 bp,其中A+T平均含量高达68.53%。序列比对结果及系统进化关系显示,16只蝇类COⅠ基因的分子鉴定与形态学鉴定结果一致。结论应用DNA条形码技术可成功扩增蝇类的COⅠ基因,其分子鉴定与形态学鉴定结果完全一致,尤其适用于微量组织样本的鉴定。Objective To establish a DNA barcoding technique for the molecular identification of flies based on CO Ⅰ gene and to make up for the deficiency of morphological identification of flies. Methods Sixteen flies commonly found at Yantai Port were used as subjects in this study. After morphological identification, total DNA was extracted from the single hind foot of each fly. According to reference, the international CO Ⅰ primer LCO1490： 5＇ -GGT CAA CAA ATC ATA AAG ATA TTG G-3＇ and HCO2198： 5＇ -TAA ACT TCA GGG TGA CCA AAA AAT CA-3＇ were used to set up a PCR system. Sequence of CO Ⅰ was successfully amplified, followed by sequencing and aligning of purified PCR product. Phylogenetic study of CO Ⅰ gene was performed using Mega 5.05 by neighbor-joining （NJ）, maximum parsimony （MP）, and maximum likelihood （ML） methods. Results The amplified CO Ⅰ fragments of sixteen flies were 690 bp in size, with an average A -4- T content of 68.53%. The sequence alignment and phylogenetic study showed that the results of CO Ⅰ gene-based identification were consistent with morphological identification. Conclusion The DNA barcoding technique established in this study is able to amplify CO Ⅰ gene from flies and thereby identify flies with the same result as morphological identification; this technique is particularly suitable for the identification of a trace amount of samples.

关 键 词：DNA条形码 细胞色素C氧化酶亚基Ⅰ基因 蝇类 分类鉴定 

分 类 号：R384.2[医药卫生—医学寄生虫学]