首个仁果类果树主要病毒RT-PCR检测试剂盒的研制
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摘要
以GF305、弗吉尼亚小苹果为带毒(阳性)和非带毒(阴性)试材,利用作者已建立的总RNA提取方法提取试材中的总RNA,根据三种病毒(ACLSV,ASGV和ASPV)外壳蛋白基因序列设计寡核苷酸引物,采用RT-PCR技术研制三种病毒检测试剂盒,达到最优化检测。并应用该试剂盒对果园中存在的病毒进行了检测。该试剂盒检测病毒速度快(6~7 h)、灵敏度高(较ELISA法高8 000倍)、专一性强、检测成本较低,是一种有推广价值的仁果类果树主要病毒检测方法。
GF305 and Virginia small apples were taken as toxic(positive) and non toxic(negative) samples,and the total RNA of the samples were extracted by the established total RNA extraction method.Oligonucleotide primers were designed according to the coat protein gene sequences of three viruses(ACLSV,ASGV and ASPV).RT-PCR technology was used to develop three kinds of virus detection kit to achieve the optimal detection,which were applied to detect the virus in orchard.The results showed that this virus detection kit had faster speed(6~7 h)and higher sensitivity(8 000 times higher than ELISA),stronger specificity,and lower detection cost,which could be the main virus detection method of pome fruit trees with promotion value.
GF305 and Virginia small apples were taken as toxic(positive) and non toxic(negative) samples,and the total RNA of the samples were extracted by the established total RNA extraction method.Oligonucleotide primers were designed according to the coat protein gene sequences of three viruses(ACLSV,ASGV and ASPV).RT-PCR technology was used to develop three kinds of virus detection kit to achieve the optimal detection,which were applied to detect the virus in orchard.The results showed that this virus detection kit had faster speed(6~7 h)and higher sensitivity(8 000 times higher than ELISA),stronger specificity,and lower detection cost,which could be the main virus detection method of pome fruit trees with promotion value.
引文
Hou Y.L.,Zhang K.C.,Hu W.Y.,Wu L.P.,Lin K.,and Yin S.P.,2002a,Detection of several RNA viruses in fruit trees by RT-PCR,Bingdu Xuebao(Chinese Journal of Virology),18(1):71-73(侯义龙,张开春,胡文玉,吴禄平,林柯,尹淑萍,2002a,逆转录-聚合酶链式反应检测果树RNA病毒,病毒学报,18(1):71-73)
Hou Y.L.,Zhang K.C.,and Yang J.L.,2005,Detection of PNRSV and PDV in peach and cherry by RT-PCR,Guoshu Xuebao(Journal of Fruit Science),22(3):292-293(侯义龙,张开春,杨俊玲,2005,应用RT-PCR方法检测桃和樱桃及其组培苗上的PNRSV和PDV,果树学报,22(3):292-293)
Hou Y.L.,Zhang K.C.,Wu L.P.,Hu W.Y.,Lin K.,Feng X.Y.,and Xing F.Y.,2002b,New methods for total RNA extraction in fruit trees,Shenyang Nongye Daxue Xuebao(Journal of Shenyang Agricultural University),33(2):122-125(侯义龙,张开春,吴禄平,胡文玉,林珂,冯晓元,邢福有,2002b,果树组织总RNA提取新方法,沈阳农业大学学报,33(2):122-125)
Menzel W.,Jelkmann W.,and Maiss E.,2002,Detection of four apple viruses bymultiplex RT-PCR assays with coamplification of plant mRNA as internal control,Journal of Virological Methods,99(1-2):81-92
Silva F.N.,Nickel O.,Fajardo T.V.M.,and Bogo A.,2008,Biological multiple indexing and RT-PCR detection of latent viruses in apple plants,Tropical Plant Pathology,33(2):157-161
Watpade S.,Raigond B.,Pramanick K.K.,Sharma N.,Handa A.,and Sarma U.,2013,Simultaneous detection of apple chlorotic leaf spot virus and apple mosaic virus in crab apples and apple rootstocks by duplex RT-PCR,Scientia Horticulturae,164:88-97
Wu Y.Q.,Chen S.Y.,Wang W.H.,and Wang X.F.,1998,Comparision of three ELISA method for detection of apple chloritic leaf spot virus and apple stem grooving virus,Zhiwu Baohu Xuebao(Acta Phytophylacica Sinica),25(3):245-248(吴雅琴,陈霜莹,王文慧,王小凤,1998,三种ELISA方法检测苹果褪绿叶斑病毒和苹果茎沟病毒的比较,植物保护学报,25(3):245-248)
Hou Y.L.,Zhang K.C.,and Yang J.L.,2005,Detection of PNRSV and PDV in peach and cherry by RT-PCR,Guoshu Xuebao(Journal of Fruit Science),22(3):292-293(侯义龙,张开春,杨俊玲,2005,应用RT-PCR方法检测桃和樱桃及其组培苗上的PNRSV和PDV,果树学报,22(3):292-293)
Hou Y.L.,Zhang K.C.,Wu L.P.,Hu W.Y.,Lin K.,Feng X.Y.,and Xing F.Y.,2002b,New methods for total RNA extraction in fruit trees,Shenyang Nongye Daxue Xuebao(Journal of Shenyang Agricultural University),33(2):122-125(侯义龙,张开春,吴禄平,胡文玉,林珂,冯晓元,邢福有,2002b,果树组织总RNA提取新方法,沈阳农业大学学报,33(2):122-125)
Menzel W.,Jelkmann W.,and Maiss E.,2002,Detection of four apple viruses bymultiplex RT-PCR assays with coamplification of plant mRNA as internal control,Journal of Virological Methods,99(1-2):81-92
Silva F.N.,Nickel O.,Fajardo T.V.M.,and Bogo A.,2008,Biological multiple indexing and RT-PCR detection of latent viruses in apple plants,Tropical Plant Pathology,33(2):157-161
Watpade S.,Raigond B.,Pramanick K.K.,Sharma N.,Handa A.,and Sarma U.,2013,Simultaneous detection of apple chlorotic leaf spot virus and apple mosaic virus in crab apples and apple rootstocks by duplex RT-PCR,Scientia Horticulturae,164:88-97
Wu Y.Q.,Chen S.Y.,Wang W.H.,and Wang X.F.,1998,Comparision of three ELISA method for detection of apple chloritic leaf spot virus and apple stem grooving virus,Zhiwu Baohu Xuebao(Acta Phytophylacica Sinica),25(3):245-248(吴雅琴,陈霜莹,王文慧,王小凤,1998,三种ELISA方法检测苹果褪绿叶斑病毒和苹果茎沟病毒的比较,植物保护学报,25(3):245-248)