应用重组酶介导核酸扩增技术快速鉴定新菠萝灰粉蚧

Rapid detection for Dysmicoccus neobrevipes by recombinase-aid amplification

本文建立了基于重组酶介导核酸扩增(RAA)技术快速鉴定新菠萝灰粉蚧(Dysmicoccus neobrevipes)反应体系,并利用该技术开展了口岸截获粉蚧的检测与鉴定。通过分析河南口岸常截获的17种粉蚧线粒体COI基因序列,设计筛选了基于RAA技术的新菠萝灰粉蚧的特异性引物,并对其进行条件优化。结果表明线粒体COI基因能准确区分新菠萝灰粉蚧与其他16种粉蚧;新菠萝灰粉蚧RAA特异性引物能稳定扩增出片段大小为323 bp单一条带,最适宜扩增温度为恒温39℃,扩增时间为20～30 min,检测DNA浓度限值为1.6×10^(-3)μg/mL。本文利用RAA技术建立的新菠萝灰粉蚧快速鉴定方法,耗时短,灵敏度高,重复性好,适用于一线口岸初筛鉴定。

This study reported a rapid method for detection of Dysmicoccus neobrevipes by recombinae-aid amplification(RAA),and the method was used to identify the mealybug intercepted at Henan ports.By analyze the mtDNA COI of 17 species of mealybugs which had been intercepted often at Henan ports,the specific primers targeting mtDNA COI of D.neobrevipes were designed and screened based on RAA technology,and the reaction condition were optimized.The results showed that D.neobrevipes was distinguished from the other 16 species of mealybugs by the mtDNA CO I.A 323 bp single band was amplified by the specific primers;the most suitable temperature and react time of RAA detection was 39℃and 20-30 minutes,respectively;the sensitivity of the primers was 1.6×10-3μg/mL.The method for detection of Dysmicoccus neobrevipes by RAA is rapid,sensitive and repeatable,and suitable for preliminary screening at port.