利用28S rDNA种特异引物鉴定新菠萝灰粉蚧
Identification of Dysmicoccus neobrevipes Beardsley (Hemiptera: Pseudococcus) using 28S rDNA species-specific primers
黄蓬英1** 林凌鸿2 方志鹏1 林玲玲1 廖富荣1
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DOI：10.7679/j.issn.2095-1353.2017.079
作者单位：1. 厦门出入境检验检疫局检验检疫技术中心，厦门 361026；2. 福建农林大学植物保护学院，福州 350002
中文关键词：新菠萝灰粉蚧，粉蚧，种特异引物，分子检测，快速鉴定
英文关键词：Dysmicoccus neobrevipes, mealybug, 28S rDNA species specific primers, molecular detection, rapid identification
中文摘要：
【目的】 新菠萝灰粉蚧Dysmicoccus neobrevipes Beardsley是近年来口岸中经常截获的检疫性有害生物，其与近似种形态差异小，极易随进境果蔬及种苗传播蔓延，但却难以快速准确鉴定。【方法】 以新菠萝灰粉蚧为靶标，进境水果中常截获的其他6种粉蚧为对照，利用扩增粉蚧的28S rDNA通用引物扩增7种粉蚧基因部分序列，同时应用Primer Premier 5软件设计新菠萝灰粉蚧28S rDNA种特异引物1对（D.neo-28SF/R）。【结果】 获得了新菠萝灰粉蚧及其他6种粉蚧的28S rDNA基因部分序列；种特异引物（D.neo-28SF/R）能稳定扩增出片段大小为235 bp条带，且该引物只对新菠萝灰粉蚧具有扩增能力，对其他6种粉蚧不具有扩增效果；对不同寄主来源新菠萝灰粉蚧亦具有同样的扩增效能。【结论】 利用基于28S rDNA种特异性PCR方法，建立了新菠萝灰粉蚧快速分子检测技术。该技术方法完全可用于新菠萝灰粉蚧的准确识别，对有效阻截其进一步扩张蔓延具有重要实践意义。
英文摘要：
[Objectives] Dysmicoccus neobrevipes Beardsley, a new invasive species in China, is a worldwide pest that poses a serious threat to agriculture and forestry. Morphological identification of this species is limited by its close similarity to other mealybug species. [Methods] To address this problem a new method for the rapid identification of D. neobrevipes was developed and a pair of 28S rDNA species-specific primers (D.neo-28SF/R) were designed. The 28S rDNA genes of D. neobrevipes and six other mealybug species were amplified and sequenced. [Results] 28S rDNA species-specific primers (D.neo-28SF/R) amplified a single band of 235 bp for D. neobrevipes. The species-specificity of this primer pair was validated with the six other mealybug species mentioned above. The results show that only D. neobrevipes rDNA was amplified, and no products from other mealybugs were obtained. The method was also tested on individual D. neobrevipes from different countries and hosts. [Conclusion] The diagnostic PCR assay established here provides a quick, simple and reliable molecular technique for the identification of D. neobrevipes, which will be useful in intercepting and preventing further spreading of this pest.