桑粉虱种特异性引物筛选

摘要

[Objective] As it is difficult to identify P.moriwith naked eye,the specific primer of Pealius mori (Takahashi) was designed and selected to provide theortical basis for its detection and identification from similar whitefly.[Method] The representative mitochondrial CO Ⅰ gene sequences of 7 species whitefly in the domestic and foreign homology with P.moriwere compared by using DNAMAN software,and five pairs of specific primers for CO [gene of P.mori were designed by using Primer 5.0 software.PCR primers were used to amplify different kinds of whitefly adults DNA with five pairs primer,and the specific bands were selected to screen the specific primers.Using the egg and larvae DNA genome as the template,the sensitivity of the selected primers were verified.[Result] Thc amplification fragments of the 2 pairs specific primers sfs1-1/sfs1-2,sfs5-1/sfs5-2 to single adult genome DNA of P.moriwere 450 bp,300 bp,respectively.But it was no amplification effect to Bemisia tabaci and Trialeurodes vaporariorum.The minimum detection value of P.mori DNA template for the 2 pairs specific primer were 0.15 pg/μl,0.15 ng/μl,respectively,and the sensitivity of primer sfs1-1/sfs1-2 was 1000 times higher than that of sfs5-1/sfs5-2.The two pairs of primers had a good ability to amplify the CO Ⅰ gene of adult,larvae and eggs of P.mori.[Conclusion] Primers of sfs1-1/sfs1-2 and sfs5-1/sfs5 have the species specificity for P.mori.,and primer of sfs1-1/sfs1-2 is the first choice for the detection and identification of P.mori..%[目的]桑粉虱Pealius mori(Takahashi)与其他种类粉虱形态相似,识别困难.设计、筛选桑粉虱种特异性引物,为桑粉虱检测和鉴定提供理论依据.[方法]DNAMAN软件比较与桑粉虱同源性较高的国内外具有代表性的7种粉虱线粒体COⅠ基因序列,应用Primer 5.0软件设计5对桑粉虱COⅠ基因特异性引物.将各对引物与不同种类粉虱成虫基因组DNA模板进行PCR扩增,检测目的条带,筛选桑粉虱种的特异性引物.以桑粉虱的卵和幼虫基因组DNA为模板,验证所筛选出的桑粉虱种特异性引物的灵敏性.[结果]2对桑粉虱种特异性引物sfs1-1/sfs1-2、sfs5-1/sfs5-2对桑粉虱单头成虫基因组DNA的PCR扩增产物分别为450、300 bp,而对烟粉虱B型、温室白粉虱无扩增效果.2对引物对桑粉虱DNA模板浓度最低检测值分别为0.15 pg/μ l、0.15 ng/μl.引物sfs1-1/sfs1-2灵敏性较引物sfs5-1/sfs5-2高1000倍.2对引物对桑粉虱成虫、幼虫及卵粒的线粒体CO Ⅰ基因皆具较好扩增能力.[结论]引物sfs1-1/sfs1-2、sfs5-1/sfs5具桑粉虱种的特异性,引物sfs1-1/sfs1-2为桑粉虱检测、鉴定的首选引物.