甘薯羽状斑驳病毒的RT-PCR检测技术构建
|
摘要
随着现代分子生物学技术的发展,反转录聚合酶链式反应技术(reverse transcription-polymerase chain reaction, RT-PCR)在甘薯病毒检测上的应用越来越广泛,其有灵敏度高、特异性强等优点,采用该技术可检测出甘薯组织中含量极低的病毒RNA。甘薯羽状斑驳病毒(sweet potato feathery mottle virus, SPFMV)是导致甘薯发生病害的主要病毒之一,为建立甘薯SPFMV快速检测技术,更好地防止该病毒的发生,本研究根据NCBI GenBank中收录的甘薯羽状斑驳病毒外壳蛋白(CP)基因核苷酸序列的保守区域设计了4对特异性引物,使用天根生化科技(北京)有限公司生产的植物总RNA提取试剂盒、FastQuant cDNA第一链合成试剂盒和2×Taq PCR MasterMix试剂盒,完成特异性引物的筛选并设置不同的退火温度对PCR扩增体系进行优化,确定了甘薯羽状斑驳病毒的RT-PCR检测技术体系。该方法的建立旨在为生产上甘薯SPFMV病毒的快速检测提供技术支持。
With the development of modern molecular biological technology, reverse transcription-polymerase chain reaction has been widely used in the detection of sweet potato viruses. Little RNA viruses from sweet potato tissue can be detected by RT-PCR which is with such advantage as more sensitive and specificity. SPFMV is one of the main viruses which reduce in sweet potato viruses. In order to build a rapid detection technology and better prevent the occurrence of the virus, four primers were designed and synthesized according to the included nucleotide sequence of coat protein gene of sweet potato feather mottle virus(SPFMV) in NCBI GenBank. The PCR amplification system was optimized by selecting specific primers and setting different annealing temperatures using the total RNA extraction kit, FastQuant cDNA first strand synthesis kit and 2 ×Taq PCR MasterMix kit produced by tiangen biochemical technology(Beijing) Co. LTD. The RT-PCR detection system for the PCR virus was determined. The method was established to provide technical support for the rapid detection of SPFMV virus in sweet potato.
With the development of modern molecular biological technology, reverse transcription-polymerase chain reaction has been widely used in the detection of sweet potato viruses. Little RNA viruses from sweet potato tissue can be detected by RT-PCR which is with such advantage as more sensitive and specificity. SPFMV is one of the main viruses which reduce in sweet potato viruses. In order to build a rapid detection technology and better prevent the occurrence of the virus, four primers were designed and synthesized according to the included nucleotide sequence of coat protein gene of sweet potato feather mottle virus(SPFMV) in NCBI GenBank. The PCR amplification system was optimized by selecting specific primers and setting different annealing temperatures using the total RNA extraction kit, FastQuant cDNA first strand synthesis kit and 2 ×Taq PCR MasterMix kit produced by tiangen biochemical technology(Beijing) Co. LTD. The RT-PCR detection system for the PCR virus was determined. The method was established to provide technical support for the rapid detection of SPFMV virus in sweet potato.
引文
He H.W.,He H.Y.,Tan G.N.,Liu Y.M.,He X.M.,Tang Z.P.,Li L.S.,and Wang H.,2014,Detection of SPFMV and SPVGby using reverse dot blot hybridization system,Nanfang N-ongye Xuebao(Journal of Southern Agriculture),45(1):43-48(何海旺,何虎翼,谭冠宁,刘义明,何新民,唐洲萍,李丽淑,王晖,2014,反向斑点杂交法快速检测甘薯羽状斑驳病毒和甘薯G病毒,南方农业学报,45(1):43-48)
Karyeija R.F.,Kreuze J.F.,Gibson R.W.,and Valkonen J.P.,2000,Synergistic interactions of a potyvirus and a phloemlimited crinivirus in sweet potato plants,Virology,269(1):26-36
Laurie S.M.,Thompson G.J.,and van den Berg A.A.,2001,Present state of quality and yield of South African sweet potato cultivars,In:Nakazawa Y.,and Ishiguro K.(eds.),Kyushu National Agricultural Experiment Station,Miyakonojo,Miyazaki,Japan,pp.55-61
Li H.W.,Xu Y.Q.,and Qiu S.H.,2015,Establishment and appli cation of a multiplex RT-PCR detection method for SPCSV,SPVG and SPFMV infecting sweet potato,Henongxue Bao(Journal of Nuclear Agricultural Science),29(8):1464-1470(李华伟,许泳清,邱思鑫,2015,侵染甘薯的SPCSV,SPVG,SPFMV多重RT-PCR检测方法的建立及应用,核农学报,29(8):1464-1470)
Ma D.F.,Li H.M.,Zhang D.P.,and Yi W.,2001,Sweet potato varieties decline in China and the prevent practices,In:Nakazawa Y.,and Ishiguro K.(eds.),International workshop on sweet potato cultivar decline study,Kyushu National Agricultural Experiment Station,Miyakonojo,Miyazaki,Japan,pp.20-26
Mori M.,Sakai J.,Kimura T.,Usugi T.,Hayashi T.,Hanada K.,and Nishiguchi M.,1995,Nucleotide sequence analysis of two nuclear inclusion body and coat protein genes of a sweet potato feathery mottle virus severe strain(SPFMV-S)genomic RNA,Arch.Virol.,140(8):1473-1482
Sakai J.,2001,Identification of four SPFMV strains and SPVGby RT-PCR and RFLP methods,In:Nakazawa Y.,and Ishiguro K.(eds.),International workshop on sweet potato cultivar decline study,Kyushu National Agricultural Experiment Station,Miyakonojo,Miyazaki,Japan,pp.85-89
Salazar L.F.,and Fuentes S.,2001,Current knowledge on major virus diseases of sweet potatoes,In:Nakazawa Y.,and Ishiguro K.(eds.),International workshop on sweet potato cultivar decline study,Kyushu National Agricultural Experiment Station,Miyakonojo,Miyazaki,Japan,pp.14-19
Shan L.N.,Ge Y.L.,Li J.B.,and Zheng C.J.,2001,Advance of aetiological study on sweet potato viruses,Henan Nongye Daxue Xuebao(Journal of Henan Agricultural University),35(1):92-96(单林娜,葛应兰,李建波,郑翠娟,2001,甘薯病毒病原学研究进展,河南农业大学学报,35(1):92-96)
Tan S.L.,2001,Problems related to yield performance decline in sweet potato in Malaysia,In:Nakazawa Y.,and Ishiguro K.(eds.),International workshop on sweet potato cultivar decline study,Kyushu National Agricultural Experiment Station,Miyakonojo,Miyazaki,Japan,pp.27-32
Wang L.,Wang Z.D.,Qiao Q.,Qin Y.H.,Zhang D.S.,Tian Y.T.,Wang S.,Zhang L.J.,and Zhang Z.C.,2013,Development of real-time fluorescent quantitative PCR assay for detection of sweet potato feathery mottle virus,Shenyang Nongye Daxue Xuebao(Journal of Shenyang Agricultural University),44(2):129-135(王丽,王振东,乔奇,秦艳红,张德胜,田雨婷,王爽,张立军,张振臣,2013,甘薯羽状斑驳病毒实时荧光定量PCR检测方法的建立,沈阳农业大学学报,44(2):129-135)
Xu Y.Q.,Li H.W.,Qiu S.X.,Liu Z.H.,Qiu Y.X.,Luo W.B.,Ji R.C.,Tang H.,and Yu H.,2014,Development of dutiplex RT-PCR for the detection of SPFMV and SPCSV in sweet potato,Fujian Nongye Xuebao(Fujian Journal of Agricultural Sciences),29(11):1114-1117(许泳清,李华伟,邱思鑫,刘中华,邱永祥,罗文彬,纪荣昌,汤浩,余华,2014,甘薯羽状斑驳病毒和褪绿矮化病毒双重RT-PCR检测方法的建立,福建农业学报,29(11):1114-1117)
Zhang Z.C.,Ma H.Q.,and Zhang G.L.,2000,Advance in virus diseases of sweet potato,Henan Nongye Kexue(Journal of Henan Agricultural Sciences),29(9):19-22(张振臣,马淮琴,张桂兰,2000,甘薯病毒病研究进展,河南农业科学,29(9):19-22)
Zhao D.L.,Tang J.,Zhang Y.G.,Shi X.M.,and Zhou Z.L.,2007,Detection of sweet potato feathery mottle virus using RT-PCR,Xibei Nongye Xueybao(Acta Agriculturae BorealiOccidentalis Sinica),16(6):199-202(赵冬兰,唐君,张允刚,史新敏,周志林,2007,甘薯羽状斑驳病毒RT-PCR检测,西北农业学报,16(6):199-202)
Karyeija R.F.,Kreuze J.F.,Gibson R.W.,and Valkonen J.P.,2000,Synergistic interactions of a potyvirus and a phloemlimited crinivirus in sweet potato plants,Virology,269(1):26-36
Laurie S.M.,Thompson G.J.,and van den Berg A.A.,2001,Present state of quality and yield of South African sweet potato cultivars,In:Nakazawa Y.,and Ishiguro K.(eds.),Kyushu National Agricultural Experiment Station,Miyakonojo,Miyazaki,Japan,pp.55-61
Li H.W.,Xu Y.Q.,and Qiu S.H.,2015,Establishment and appli cation of a multiplex RT-PCR detection method for SPCSV,SPVG and SPFMV infecting sweet potato,Henongxue Bao(Journal of Nuclear Agricultural Science),29(8):1464-1470(李华伟,许泳清,邱思鑫,2015,侵染甘薯的SPCSV,SPVG,SPFMV多重RT-PCR检测方法的建立及应用,核农学报,29(8):1464-1470)
Ma D.F.,Li H.M.,Zhang D.P.,and Yi W.,2001,Sweet potato varieties decline in China and the prevent practices,In:Nakazawa Y.,and Ishiguro K.(eds.),International workshop on sweet potato cultivar decline study,Kyushu National Agricultural Experiment Station,Miyakonojo,Miyazaki,Japan,pp.20-26
Mori M.,Sakai J.,Kimura T.,Usugi T.,Hayashi T.,Hanada K.,and Nishiguchi M.,1995,Nucleotide sequence analysis of two nuclear inclusion body and coat protein genes of a sweet potato feathery mottle virus severe strain(SPFMV-S)genomic RNA,Arch.Virol.,140(8):1473-1482
Sakai J.,2001,Identification of four SPFMV strains and SPVGby RT-PCR and RFLP methods,In:Nakazawa Y.,and Ishiguro K.(eds.),International workshop on sweet potato cultivar decline study,Kyushu National Agricultural Experiment Station,Miyakonojo,Miyazaki,Japan,pp.85-89
Salazar L.F.,and Fuentes S.,2001,Current knowledge on major virus diseases of sweet potatoes,In:Nakazawa Y.,and Ishiguro K.(eds.),International workshop on sweet potato cultivar decline study,Kyushu National Agricultural Experiment Station,Miyakonojo,Miyazaki,Japan,pp.14-19
Shan L.N.,Ge Y.L.,Li J.B.,and Zheng C.J.,2001,Advance of aetiological study on sweet potato viruses,Henan Nongye Daxue Xuebao(Journal of Henan Agricultural University),35(1):92-96(单林娜,葛应兰,李建波,郑翠娟,2001,甘薯病毒病原学研究进展,河南农业大学学报,35(1):92-96)
Tan S.L.,2001,Problems related to yield performance decline in sweet potato in Malaysia,In:Nakazawa Y.,and Ishiguro K.(eds.),International workshop on sweet potato cultivar decline study,Kyushu National Agricultural Experiment Station,Miyakonojo,Miyazaki,Japan,pp.27-32
Wang L.,Wang Z.D.,Qiao Q.,Qin Y.H.,Zhang D.S.,Tian Y.T.,Wang S.,Zhang L.J.,and Zhang Z.C.,2013,Development of real-time fluorescent quantitative PCR assay for detection of sweet potato feathery mottle virus,Shenyang Nongye Daxue Xuebao(Journal of Shenyang Agricultural University),44(2):129-135(王丽,王振东,乔奇,秦艳红,张德胜,田雨婷,王爽,张立军,张振臣,2013,甘薯羽状斑驳病毒实时荧光定量PCR检测方法的建立,沈阳农业大学学报,44(2):129-135)
Xu Y.Q.,Li H.W.,Qiu S.X.,Liu Z.H.,Qiu Y.X.,Luo W.B.,Ji R.C.,Tang H.,and Yu H.,2014,Development of dutiplex RT-PCR for the detection of SPFMV and SPCSV in sweet potato,Fujian Nongye Xuebao(Fujian Journal of Agricultural Sciences),29(11):1114-1117(许泳清,李华伟,邱思鑫,刘中华,邱永祥,罗文彬,纪荣昌,汤浩,余华,2014,甘薯羽状斑驳病毒和褪绿矮化病毒双重RT-PCR检测方法的建立,福建农业学报,29(11):1114-1117)
Zhang Z.C.,Ma H.Q.,and Zhang G.L.,2000,Advance in virus diseases of sweet potato,Henan Nongye Kexue(Journal of Henan Agricultural Sciences),29(9):19-22(张振臣,马淮琴,张桂兰,2000,甘薯病毒病研究进展,河南农业科学,29(9):19-22)
Zhao D.L.,Tang J.,Zhang Y.G.,Shi X.M.,and Zhou Z.L.,2007,Detection of sweet potato feathery mottle virus using RT-PCR,Xibei Nongye Xueybao(Acta Agriculturae BorealiOccidentalis Sinica),16(6):199-202(赵冬兰,唐君,张允刚,史新敏,周志林,2007,甘薯羽状斑驳病毒RT-PCR检测,西北农业学报,16(6):199-202)