美洲斑潜蝇SS-PCR检测技术研究

【背景】美洲斑潜蝇是一种严重威胁瓜果蔬菜、烟草、棉花等经济作物和花卉生产的入侵性害虫。由于潜叶蝇类害虫体型较小、生活方式隐蔽、形态相似,本文针对其难以快速准确地进行形态鉴别的问题,以美洲斑潜蝇为研究对象,以菜田常见的4种潜叶蝇类害虫为参照,采用种特异性PCR方法（species-specific PCR,SS-PCR）,研究其快速分子检测鉴定技术。【方法】调用GenBank中一段936bp的美洲斑潜蝇线粒体DNA（mtDNA）细胞色素氧化酶亚基Ⅰ基因（COⅠ）的序列（Gen-Bank登录号为EU219613）,并根据此基因片段的碱基序列设计引物1对,其扩增片段大小为294bp。【结果】种特异性检验结果显示,该引物只对美洲斑潜蝇的COⅠ基因具有扩增能力,对其他种类如南美斑潜蝇、三叶斑潜蝇、葱斑潜蝇、豌豆潜叶蝇等没有扩增能力。该引物不仅对成虫具有良好的扩增效果,对蛹、幼虫以及单粒卵也具有同样的扩增效果,其最低检出阈值为1/3840头成虫。【结论与意义】SS-PCR技术体系可用于美洲斑潜蝇的鉴定识别与检测监测,对阻止其进一步扩散蔓延具有重要意义。

Species-specific PCR primers for identification of Liriomyza sativae Blanchard

Background] Liriomyza sativae Blanchard（Diptera：Agromyzidae）,an invasive alien species,is an important pest on many vegetables,flowers,tobacco,and cotton in many agricultural areas in China.Morphological identification of L.sativae is limited by small size,the high degree of similarity to related species and polymorphism.In this study,a method was described for the development of DNA marker for the identification of L.sativae.Method]A pair of species-specific PCR（SS-PCR） primers based on a fragment of known mitochondrial DNA cytochrome oxidase I（mtDNA CO Ⅰ） sequence（936 bp,GenBank accession no.：EU219613） was designed for L.sativae.Result] The SS-PCR primers amplified a single band of 294 bp of L.sativae.The specificity of the primers was validated using four other leafminer species,including Liriomyza huidobrensis（Blanchard）,Liriomyza trifolli（Brugess）,Liriomyza chinensis（Kato） and Phytomyza horticola Goureau,all of them commonly occurring in China.All L.sativae specimens were correctly identified,and no cross-reactions with other leafminer species were observed.The method was tested on single individuals in the egg,first-,second-and third-instar larvae,pupa and adult（male and female） stages,and proved to be applicable for all life stages.Moreover,the 294 bp mtDNA fragment could be clearly identified even at the dilution as low as 1/3840 of a whole female adult of L.sativae.Conclusion and significance] The SS-PCR method developed is promising,effective,fast and economic identification,hence proposed as a valuable alternative to traditional identification of this insect species.This technique should be applicable in quarantine testing for L.sativae during transportation of seedlings of flowers and vegetables and also in monitoring and management of this insect pest.