摘要
针对粉虱类害虫难以准确快速地进行形态鉴别的问题,以局部发生的黑刺粉虱Aleurocanthus spiniferus(Quaintance)为对象,采用特征序列扩增区域(SCAR)标记法,研究其快速分子检测技术。利用SCAR标记技术获得了长度为987bp的黑刺粉虱特异性片段(GenBank登录号为FJ613323),根据此片段的碱基序列设计黑刺粉虱特异性引物1对(AS-F518/AS-R938),其扩增片段为421bp。种特异性检验结果显示,该对引物只对黑刺粉虱的基因组具有扩增能力,对同域发生的桔绿粉虱Dialeurodes citri(Ashmead)以及其他种类的粉虱如烟粉虱Bemisia tabaci(Gennadius)B型、Q型、ZHJ-1型和ZHJ-2型,温室粉虱Trialeurodes vaporariorum(Westwood)以及螺旋粉虱Aleurodicus disperses(Russell)等的基因组不具有扩增效果。该引物不仅对成虫具有良好的扩增效能,对卵、2龄若虫和拟蛹等亦具有同样的扩增能力,其最低检出限为1/1920头成虫。该技术体系的建立在茶树和柑桔苗木调运的害虫检疫和监测/检测中具有重要意义。
Morphological identification of whitefly is limited by small size, the high degree of similarity and polymorphism. In this study, the method for the development of DNA markers for rapid detection of the spiny whitefly, Aleurocanthus spiniferus （Quaintance）, was developed based on sequence characterized amplified region （SCAR） derived from a randomly amplified polymorphic DNA （RAPD） band. An approximately 990 bp DNA fragment of A. spiniferus, species [e. g., Dialeurodes citri （Ashmead） , Bemisia which is absent in other closely related whitefly tabaci （Gennadius） （biotvnes B, Q, ZHJ-1 and ZHJ-2）, Trialeurodes vaporariorum （Westwood） and Aleurodicus disperses （ RAPD-PCR analysis. After cloning and sequencing the target fragment （987 Russell） ], was identified by bp, GenBank accession no. ： FJ613323）, one pair of SCAR primers （AS-F518/AS-R938） was developed, which could amplify a single fragment of 421 bp in length. Specificity tests performed with this pair of primers showed that all A.spiniferus specimens were detectable and no cross reactions with other whitefly species were observed. The method was tested on egg （3 eggs as one sample） , single 2nd and 4th instar nymph, and male and female adults, and proved to be applicable for these stages of A. spiniferus. Moreover, the 421 bp DNA fragment could be clearly identified even at the dilution as low as 1/1 920 of a whole female adult of A. spiniferus for all replicates. This technique should be useful in quarantine and detection of A. spiniferus during transportation of the seedlings of tea and orange.
出处
《昆虫学报》
CAS
CSCD
北大核心
2009年第8期895-900,共6页
Acta Entomologica Sinica
基金
国家“973”计划项目(2009CB119200)
国家科技支撑计划项目(2006BAD08A14)
公益性行业(农业)科研专项(200803005)
国家自然科学基金项目(30571254)
中央财政国家重点实验室自主研究课题专项经费(SKL2007SR04)
关键词
黑刺粉虱
RAPD
SCAR
快速鉴定
特异性扩增
Aleurocanthus spiniferus
RAPD
SCAR
rapid identification
specific amplification
