三叶草斑潜蝇DNA条形码鉴定和ELISA检测的方法学比较

摘要

[Purpose] Study on the capability of DNA barcoding based on COI and ITS1 sequences and indirect ELISA on identi6cation Liriomyza trifolii. [Method] (1) COI and ITS1 sequences were amplified using the DNA extracted from L. trifolii raised in lab, and compared with COI and ITS1 sequences of L. trifolii, L. sativae, L. huidobrenisis and Chromatomyia horticola from Genbank after sequencing and assembling. The genetic analysis was performed. (2) Based on the indirect ELISA method established in our lab, the reaction of the larval, pupa and adult of L. huidobrenisis and C. horticola with the monoclonal antibody against L. trifolii were detected respectively. [Result] (1) L. trifolii can be identified with DNA barcoding based on COI and ITS1 sequences. For COI sequence, the genetic similarity between L. trifolii and L. huidobrenisis was 0.68, which was the highest among 4 species of leafminer. For ITS1 sequence, the genetic similarity between L trifolii and L. sativae was the hightest. It was 0.885. (2)Only L. trifolii had a positive reaction with the monoclonal antibody against L. trifolii test by indirect ELISA. [Conclusion] Both DNA barcoding and indirect ELISA can be used to identify the L. trifolii. The method of DNA barcoding based on COI sequence shows higher confidence than that based on ITS1 sequence. ELISA is low cost and simple for agricultural technicians to use. The ELISA method can deal with hundreds or even thousands of samples at the same time. It is more suitable for the detection in a large number of field samples.%[目的]探讨以COI和ITS1序列为条形码和间接ELISA方法鉴定三叶草斑潜蝇的可行性.[方法]①提取实验室饲养的三叶草斑潜蝇基因组DNA,扩增了三叶草斑潜蝇细胞色素C氧化酶亚基I(COI)和内转录第一间隔区(ITS1)序列,经测序、拼接后,与Genbank中三叶草斑潜蝇、美洲斑潜蝇、南美斑潜蝇及豌豆彩潜蝇的COI及ITS1序列进行比对,并进行遗传分析.②基于本实验室建立的ELISA检测方法,分别检测了三叶草斑潜蝇单克隆抗体与南美斑潜蝇及豌豆彩潜蝇的幼虫、蛹和成虫的交叉反应情况.[结果](COI和ITS1序列均可以将三叶草斑潜蝇同其它3种近缘种区分开.对于COI序列,三叶草斑潜蝇与南美斑潜蝇相似度为68％,亲缘关系最近；对于ITS1序列,三叶草斑潜蝇与美洲斑潜蝇相似度为88.5％,亲缘关系最近.②间接ELISA检测只有三叶草斑潜蝇呈阳性反应,与南美斑潜蝇及豌豆彩潜蝇幼虫、蛹和成虫均不发生交叉反应.[结论]以COI或ITS1序列为条形码的DNA鉴定法和以单克隆抗体为基础的ELISA检测均可区分三叶草斑潜蝇与其它3种近缘种.其中COI序列作为条形码鉴定可信度优于ITS1序列,ELISA方法操作简单,成本低,更适于田间大量样品的鉴定.