类病毒检测技术培训于德才.ppt

样品采自大庆地区，样品为克新13号,二、类病毒种类,已知序列类病毒种类及其分类Thevariantsofviroidknownsequenceandtheirclassification,类病毒结构示意图Thestructureofviroid,马铃薯纺锤块茎类病毒热变性过程Heat-denaturedprocessdiagramofPSTVd,三、类病毒结构,马铃薯纺锤块茎类病毒的五个功能区ThefivefunctionaldomainsofPSTVd,马铃薯类病毒中国分离株核苷酸序列,251113591CCTGCACGTCGACGATTGGAGCTCACAGGAACCACGAGTTTAGTTCCGAGSacP(25-11)51GAACCAACTGCGGTTCCAAGGGCTAAACACCCTCGCCCCGAAGCAAAGAA101AGATAGAGAAAAAGCGGTTCTCGGGAGCTTCAGTTGTTTCCACCGGGTAG151TAGCCGAAGCGACAGCGCAAAGGGGGCGAGGGGTGGTCCTGCGGGCGCGA201GGAAGGACACCCGAAGAAAGGAAGGGTGAAAACCCTGTTTCGGCGGGAAT251TACTCCTGTCGGCCGCTGGGCACTCCCCACCGTCCTTATTGCCAGTTCGC301TCCAGGTTTCCCCGGGGATCCCTGAAGCGCTCCTCCGAGCCGCCTTCTTT3526351TTTCTTTTCTGCTCAGGAGGTCAGGTGTGAACGGGTACCCGATCTCTAGAP(26-35)Kpn401GGATCCCCGGGTACCGAGCTCGAATTCGTAATCATGGTCATAGCTGTTTC451CTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGA501AGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATT551AATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCC601AGCTGCATTAATGAATCGGCCAACGCGCCGGGAGAGGCGGGTTGCGTATT651T,序列测定结果,PSTVd致病区的位置和结构。图解表示PSTVd-Intermediate的完整结构，包括左未端区（TL,1-46/315-359），致病区（47-73/286-314），中央保守区（74-120/240-285），可变区（121-148/212-239）和右未端区（TR,149-211）(Keeseandsymons,1985),premelting(PM)区1-3（Steger等，1984）和二级发夹结构1-111。下面为三个自然发生的PSTVd分离株系。水平箭头指出PM1和VM区域（Schnlzer等，1985），“星号”表示中间株系和强、弱系序列差异。此图引自Owensetal(1996)。,TLPath1(M)CGGAACUAAACUCGUGGUUCCUGUGGUUCACACCUGACCUCCUGAGCAGAAAAGAAAAAA2(M)CGGAACUAAACUCGUGGUUCCUGUGGUUCACACCUGACCUCCUGAGCAGAAAAGAAAAAA3(M)CGGAACUAAACUCGUGGUUCCUGUGGUUCACACCUGACCUCCUGAGCAGAAAAGAAAAAA4(I)CGGAACUAAACUCGUGGUUCCUGUGGUUCACACCUGACCUCCUGAGCAGAAAAGAAAAAA5(S)CGGAACUAAACUCGUGGUUCCUGUGGUUCACACCUGACCUCCUGAGCAAGAAAAGAAAAAA6(S)CGGAACUAAACUCGUGGUUCCUGUGGUUCACACCUGACCUCCUGAGCAGAAAAGAAAAAACCR1(M)GAAGGCGGCUCGGAGGAGCGCUUCAGGGAUCCCCCGGGAAACCUGGAGCGAACUGGCAAU2(M)GAAGGCGGCUCGGAGGAGCGCUUCAGGGAUCCCCCGGGAAACCUGGAGCGAACUGGCAAU3(M)GAAGGCGGCUCGGAGGAGCGCUUCAGGGAUCCCCCGGGAAACCUGGAGCGAACUGGCAAU4(I)GAAGGCGGCUCGGAGGAGCGCUUCAGGGAUCCCCCGGGAAACCUGGAGCGAACUGGCAAAA5(S)GAAGGCGGCUCGGAGGAGCGCUUCAGGGAUCCCCCGGGAAACCUGGAGCGAACUGGCAAU6(S)GAAGGCGGCUCGGAGGAGCGCUUCAGGGAUCCCCCGGGAAACCUGGAGCGAACUGGCAAAAVarTR1(M)AAGGACGGUGGGGAGUGCCCAGCGGCCGACAGGAGUAAUUCCCGCCGAAACAGGGUUUUC2(M)AAGGACGGUGGGGAGUGCCCAGCGGCCGACAGGAGUAAUUCCCGCCGAAACAGGGUUUUC3(M)AAGGACGGUGGGGAGUGCCCAGCGGCCGACAGGAGUAAUUCCCGCCGAAACAGGGUUUUC4(I)AAGGACGGUGGGGAGUGCCCAGCGGCCGACAGGAGUAAUUCCCGCCGAAACAGGGUUUUC5(S)AGGACGGUGGGGAGUGCCCAUGCGGCCGACAGGAGUAAUUCCCGCCGAAACAGGGUUUUC6(S)AAGGACGGUGGGGAGUGCCCAGCGGCCGACAGGAGUAAUUCCCGCCGAAACAGGGUUUUCVar1(M)ACCCUUCCUUUCUUCGGGUGUCCUUCCUCGCGCCCGCAGGACCACCCCUCGCCCCCUUUG2(M)ACCCUUCCUUUCUUCGGGUGUCCUUCCUCGCGCCCGCAGGACCACCCCUCGCCCCCUUUG3(M)ACCCUUCCUUUCUUCGGGUGUCCUUCCUCGCGCCCGCAGGACCACCCCUCGCCCCCUUUG4(I)ACCCUUCCUUUCUUCGGGUGUCCUUCCUCGCGCCCGCAGGACCACCCCUCGCCCCCUUUG5(S)ACCCUUCCUUUCUUCGGGUGUCCUUCCUCGCGCCCGCAGGACCACCCCUCG-CCCCUUUG6(S)ACCCUUCCUUUCUUCGGGUGUCCUUCCUCGCGCCCGCAGGACCACCCCUCGCCCCCUUUGCCRPath1(M)CGGUGUCGCUUCGGCUACUACCCGGUGGAAACAACUGAAGCUCCCGAGAACCGCUUUUUC2(M)CGGUGUCGCUUCAGCUACUACCCGGUGGAAACAACUGAAGCUCCCGAGAACCGCUUUUUC3(M)CGGUGUCGCUUCGGAUAUUACCCGGUGGAAACAACUGAAGCUCCCGAGAACCGCUUUUUC4(I)CGGUGUCGCUUCGGCUACUACCCGGUGGAAACAACUGAAGCUCCCGAGAACCGCUUUUUC5(S)CGGUGUCGCUUCGGCUACUACCCGGUGGAAACAACUGAAGCUCCCGAGAACCGCUUUUUC6(S)CGGUGUCGCUUCGACUACUACCCGGUGGAAACAACUGAAGCUCCCGAGAACCGCUUUUUCTL1(M)UCUAUCUUUCUUUGCUUCGGGGCGAGGGUGUUUAGCCCUUGGAACCGCAGUUGGUUCCU2(M)UCUAUCUUUCUUUGCUUCGGGGCGAGGGUGUUUAGCCCUUGGAACCGCAGUUGGUUCCU3(M)UCUAUCUUUCUUUGCUUCGGGGCGAGGGUGUUUAGCCCUUGGAACCGCAGUUGGUUCCU4(I)UCUAUCUUAC-UUGCUUCGGGGCGAGGGUGUUUAGCCCUUGGAACCGCAGUUGGUUCCU5(S)UCUAUCUUUAUUUGCUUCGGCGGCGAGGGUGUUUAGCCCUUGGAACCGCAGUUGGUUCCU6(S)UCUAUCUU-AUGUGCUUCAGGGCGAGGGUGUUUAGCCCUUGGAACCGCAGUUGGUUCCU,PSTVd中国分离株与已报道序列在一级结构上的差异,PSTVd强、弱系及中间株系的核苷酸序列差异比较ComparisonwithmildstrainfromChinainnucleotidesequenceoftheisolatesofthemild,intermediateandseverestrainofpotatospindletuberviroid(PSTVd),注：FromHeroldetal.(1992);FromOwensetal.(1992);FromSinghetal.(1993);FromGrossetal.(1981);andFromGora-Sochacka(1997).,四、类病毒鉴定,1、指示植物接种鉴定1）鲁特格番茄2）新莨菪2、电泳1）垂直双向丙烯酰胺电泳（2-D-PAGE）2）往返丙烯酰胺电泳（R-PAGE）3、反转录PCR（RT-PCR）4、核酸斑点杂交（NASH）,往返丙烯酰胺电泳（R-PAGE）检测流程图,核酸提取,第一项电泳,反向电泳,固定,银染色,显色,结果判读,PSTVd,上样量：8g,1.6g,0.32g,64ng,13ng,2.6ng,520pg,104pg最低检出率：13ng,双向电泳检测PSTVd,PSTVd,检测结果：样品含有PSTVd，箭头所指为类病毒核酸带,RT-PCR检测类病毒流程,引物设计,扩增产物分析（）,以cDN为模板扩增,制备cDN第一链（反转录）,样品核酸提取,检测结果：PCR反应退火温度分别设定为55和57，在57下特异性好，可用来检测PSTVd,434bp328bp,12345,资料检索、收集、样品采集,类病毒病原鉴定:三种方法：（R-PAGE、2-D-PAGE、RT-PCR）,含PSTVdpGEM-7Z质粒,利用HaeIII对重组质粒进行酶切验证,鉴定结果：含PSTVd,设计引物、对含PSTVd的pGEM-7Z质粒进行PCR扩增,利用生物素biotin-11-dUTP代替dUTP制备探针,进行核酸斑点杂交反应,NBT/BCIP化学颜色反应,Flu荧光素发光反应,试剂盒组装,生产应用,核酸斑点杂交（）检测技术流程,探针制备,终止反应,预杂交,封闭,孵育,信号产生,杂交,漂洗,点样,尼龙膜的处理,500ng50ng5ng500pg50pg5pg,1234,200pg50pg200pg50pg,NASH方法检测PSTVd专化性测试（颜色反应）,结果显示：阳性样品有斑点，阴性样品不产生斑点。反应专化性强。,核酸斑点杂交（）检测反应原理图,核酸斑点杂交（NASH）技术的研究,探针的制备,1、Hae单酶切含PSTVd单体的pGEM-7Z质粒,123,328bp,Hae/7Z空Hae/7Z-PSTVd(bp)正向插入反向插入16576576572458458561*3434434458328*369*434289320*289267289267174267174142174142102142125*801021028080,结果显示：pGEM-7Z重组质粒含有PSTVd单体，且为正向插入,2、引物筛选,根据已报导的PSTVd基因序列（Genebank登陆号AY372400）设计两对引物对7Z