螺旋粉虱SCAR标记的建立与应用

Establishment and application of a SCAR marker for rapid diagnosis of the spiraling whitefly,Aleurodicus dispersus Russell

螺旋粉虱Aleurodicus dispersus Russell是一种严重威胁果树、蔬菜和园林植物生产的入侵性害虫。由于该粉虱的卵和初孵若虫体型微小,与其它粉虱类害虫形态相似,且易于携带传播,难以快速准确鉴别,本研究采用特征序列扩增区域（SCAR）标记法,通过对目的片段的克隆与测序,设计螺旋粉虱特征片段扩增引物,并对该引物的种特异性和灵敏性进行检验。结果显示,利用SCAR标记法获得了螺旋粉虱特异性片段（632bp,GenBank登录号为HM240855）,根据此片段的碱基序列设计1对螺旋粉虱特异性引物,其扩增片段大小为307bp;该对引物只对螺旋粉虱具有扩增能力,对近缘种、属粉虱不具有扩增效果。该引物不仅对成虫具有良好的扩增效能,对单粒卵、1~3龄若虫和拟蛹等亦具有同样的扩增能力,其最低检出阈值为1/6400头成虫。从而表明,该SCAR标记技术完全可用于螺旋粉虱的鉴定识别及其检测和监测。

The spiraling whitefly,Aleurodicus dispersus Russell,is an invasive pest,which presents tremendous risk to fruit tree,vegetable and ornamental plant products in China.The egg and young nymph of the spiraling whitefly are small in size,have high degree of morphological similarity with other whitefly species and can be carried and spread easily.It is difficult to discriminate this whitefly species from others by morphological identification.In our study,a method was described for the development of DNA marker for diagnosis the spiraling whitefly based on sequence characterized amplified region（SCAR）.A DNA fragment of A.dispersus,which absent in other closely related whitefly species,was identified by RAPD-PCR analysis.After cloning and sequencing the target fragment（632bp,GenBank accession no.： HM240855）,one pair of SCAR primers was developed,which could amplify a single fragment of 307bp in length.Specificity tests performed with this pair of primers showed all A.dispersus specimens were detected,and no cross reactions with other whitefly species were observed.The method was tested and proved to be applicable for single egg,first to third stage instars,red-eyed-nymph,male and female adults of A.dispersus.Moreover,the 307bp DNA fragment could be clearly identified when the dilution was 1/6400 of an A.dispersus female adult for all replicates.This method will make it possible for on-site diagnostic testing,routine screening of seedlings of flowers,ornamentals and forests at ports of entry,facilitate monitoring of spread,prevention of new incursions,and further damage caused by A.dispersus in China.