外来入侵物种细足捷蚁的SS-PCR快速检测技术研究

Rapid identification of yellow crazy ant Anoplolepis gracilipes(Hymenoptera:Formicidae) by using species-specific PCR technology

细足捷蚁Anoplolepis gracilipes为我国南部地区新发现的外来入侵物种,对入侵地区生物多样性已造成严重威胁。本研究针对其个体小、表型特征较难鉴定等问题,建立基于SS-PCR技术的细足捷蚁快速分子检测鉴定技术体系。以细足捷蚁为阳性对照,以其他7种形态相似种和常见种为阴性对照,采用基于线粒体DNA细胞色素C氧化酶亚基Ⅰ(mt DNA COI)基因序列的种特异性PCR方法 (species-specific PCR,SS-PCR),研究其快速分子检测鉴定技术的关键指标和反应条件。利用昆虫mt DNA COI基因通用型引物LCO-1490/HCO-2198获得细足捷蚁及其他7种形态相似种和常见蚂蚁种的COI序列,根据测序比对及筛选得到1对高灵敏度引物APGPF/APGPR,其扩增片段大小为374 bp。种特异性检验结果表明,该引物只对细足捷蚁的COI基因具有扩增能力,对其他7种形态相似种和常见种包括拟光腹弓背蚁Camponotus irritans(Smith,1857)、费氏弓背蚁Camponotus fedtschenkoi(Mayr,1877)、尼科巴弓背蚁Camponotus nicobarensis(Mayr,1865)、黄猄蚁Oecophylla smaragdina(Fabricius,1775)、大阪举腹蚁Crematogaster osakensis(Forel,1900)、宽结大头蚁Pheidole noda(Smith,1874)、大头蚁Pheidole sp.不具有特异性扩增效果。灵敏性检验的结果表示,该对引物不仅对成年工蚁具有良好的扩增能力,对不同品级与龄期的蚂蚁卵、蛹、幼虫也具有同样的扩增能力,其检测阈值为22.00 pg/μL(相当于1/51 200头成虫)。建立的细足捷蚁对其他7种形态相似种和常见种的物种特异性检测鉴定技术,可用于细足捷蚁的快速识别、监测预警和控制清除等相关研究与应用。

Yellow crazy ant,Anoplolepis gracilipes,is a newly recorded invasive species in southern China,which poses a serious threat to the native biodiversity.Morphological identification of A.gracilipes is limited by small size,high degree of similarity to related species.This study aims to develop a technique based on mitochondrial DNA cytochrome c oxidase subunit I(mtDNA COI)gene sequence for the rapid identification of A.gracilipes.The fragments of mtDNA COI gene of A.gracilipes and seven other ant species were amplified and sequenced using COI gene universal primers LCO-1490/HCO-2198.One pair of species specific COI(SS-COI)primers,APGPF and APGPR,was designed.The target fragment amplified by the SS-COI primers(APGPF/APGPR)was 374 bp in length.The species specificity and sensitivity of the primer pair were tested.The species specificity of the primer pair was validated using seven other ant species,i.e.,Camponotus irritans(Smith.1857),Camponotus fedtschenkoi(Mayr.1877),Camponotus nicobarensis(Mayr.1865),Oecophylla smaragdina(Fabricius.1775),Crematogaster osakensis(Forel.1900),Pheidole noda(Smith,1874),Pheidole sp.All A.gracilipes specimens were successfully detected,and no cross reaction with other ant species was observed.The method was tested using individual worker or queen,individual 1 st instar,2 nd instar larva,and even one grain of egg,and proved to be applicable for all of these life stages and grades.Sensitivity test results demonstrated that successful amplification could be obtained with the concentration of template DNA as low as 22.00 pg/μL,equal to 1/51 200 of a whole worker of A.gracilipes.The SS-COI method developed here provides a quick,simple and reliable molecular technique for the identification and monitoring of A.gracilipes,which would be useful in detecting,monitoring and control of this new invasive ant species.