类病毒检测技术培训

1、会计学1类病毒检测技术培训类病毒检测技术培训二、类病毒种类已知序列类病毒种类及其分类 The variants of viroid known sequence and their classification中名Chinese names英名English names缩写Abbrevia-tion大小Length (nt)中央保守区Central conserved马铃薯纺锤块茎类病毒Potato spindle tuberPSTVd356-360PSTVd柑桔裂皮类病毒Citrus exocortisCEVd370-375PSTVd菊花矮化类病毒Chrysanthemum stuntCSV

2、d354-356PSTVd番茄顶矮化类病毒Tomato apical stuntTASVd360-363PSTVd番茄雄性株类病毒Tomato planta machoTPMVd360PSTVd唇膏蔓蔷薇隐性类病毒Columnea latentCLVd370-372PSTVd啤酒矮化类病毒Hop sturtHSVd297-303PSTVd椰子死亡类病毒Coconut cadang-cadangCCCVd246-247CCCVd椰子tinangaja类病毒Coconut tinangajaCTiVd254CCCVd啤酒花隐潜类病毒Hop lalentHLVd256CCCVd柑桔IV类病毒Citr

3、us IVCVdiV284CCCVd苹果锈果类病毒Apple scar skinASSVd329-330ASSVd葡萄黄斑类病毒1Grapevine yellow speckle 1GYSCd1366-368ASSVd葡萄黄斑类病毒2Grapevine yellow speckle 2GYSCd2363ASSVd澳洲葡萄类病毒Australian grapevineAGVd369ASSVd柑桔硬叶类病毒Citrus bent leafCBLVd318ASSVd梨疱溃疡类病毒Pear blister cankerPBCVd325ASSVd鳄梨白斑类病毒Avocado sunblotchASBVd

4、245-250ASBVd桃潜花叶类病毒Peach latent mosaicPLMVd336-337ASBVd锦学苏类病毒Coleus blumei ICbVd1248CbVd1 类病毒结构示意图 The structure of viroid 马铃薯纺锤块茎类病毒热变性过程 Heat-denatured process diagram of PSTVd三、类病毒结构马铃薯纺锤块茎类病毒的五个功能区 The five functional domains of PSTVd 马铃薯类病毒中国分离株核苷酸序列l 25 11 1 359l1 CCTGCACGTC GACGATTGGA GCTC AC

5、AGGA ACCACGAGTT TAGTTCCGAGl Sac P(25-11)l51 GAACCAACTG CGGTTCCAAG GGCTAAACAC CCTCGCCCCG AAGCAAAGAAl101 AGATAGAGAA AAAGCGGTTC TCGGGAGCTT CAGTTGTTTC CACCGGGTAGl151 TAGCCGAAGC GACAGCGCAA AGGGGGCGAG GGGTGGTCCT GCGGGCGCGAl201 GGAAGGACAC CCGAAGAAAG GAAGGGTGAA AACCCTGTTT CGGCGGGAATl251 TACTCCTGTC GGCCGCTGG

6、G CACTCCCCAC CGTCCTTATT GCCAGTTCGCl301 TCCAGGTTTC CCCGGGGATC CCTGAAGCGC TCCTCCGAGC CGCCTTCTTT 35 26l351 TTTCTTTTCT GCTCAGGAGG TCAGGTGTGA ACGGGTACCC GATCTCTAGAl P(26-35) Kpn l401 GGATCCCCGG GTACCGAGCT CGAATTCGTA ATCATGGTCA TAGCTGTTTCl451 CTGTGTGAAA TTGTTATCCG CTCACAATTC CACACAACAT ACGAGCCGGAl501 AGCA

7、TAAAGT GTAAAGCCTG GGGTGCCTAA TGAGTGAGCT AACTCACATTl551 AATTGCGTTG CGCTCACTGC CCGCTTTCCA GTCGGGAAAC CTGTCGTGCCl601 AGCTGCATTA ATGAATCGGC CAACGCGCCG GGAGAGGCGG GTTGCGTATTl651 Tl l 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（121-148/21

8、2-239）和右未端区（TR, 149-211）(Keese and symons, 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Owens et al (1996)。 TL Path 1(M) CGGAACUAAA CUCGUGGUUC CUGUGGUUCA CACCUGACCU CCUGAGCA GA AAAGAAAAAA 2(M) CGGAACUAAA CUCGUGGUUC CUGU

9、GGUUCA CACCUGACCU CCUGAGCA GA AAAGAAAAAA3(M) CGGAACUAAA CUCGUGGUUC CUGUGGUUCA CACCUGACCU CCUGAGCA GA AAAGAAAAAA4(I) CGGAACUAAA CUCGUGGUUC CUGUGGUUCA CACCUGACCU CCUGAGCA GA AAAGAAAAAA5(S) CGGAACUAAA CUCGUGGUUC CUGUGGUUCA CACCUGACCU CCUGAGCA GA AAAGAAAAAA6(S) CGGAACUAAA CUCGUGGUUC CUGUGGUUCA CACCUGACC

10、U CCUGAGCA GA AAAGAAAAAA CCR 1(M) GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAAU2(M) GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAAU3(M) GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAAU4(I) GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG A

11、ACUGGCAAAA5(S) GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAAU 6(S) GAAGGCGGCU CGGAGGAGCG CUUCAGGGAU CCCCCGGGAA ACCUGGAGCG AACUGGCAAAA Var TR1(M) AAGGACGGUG GGGAGUGCCC A GCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC2(M) AAGGACGGUG GGGAGUGCCC A GCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUU

12、C3(M) AAGGACGGUG GGGAGUGCCC A GCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC4(I) AAGGACGGUG GGGAGUGCCC A GCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC5(S) A GGACGGUG GGGAGUGCCC AUGCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC 6(S) AAGGACGGUG GGGAGUGCCC A GCGGCCGAC AGGAGUAAUU CCCGCCGAAA CAGGGUUUUC Var 1(M) ACCC

13、UUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUUUG2(M) ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUUUG3(M) ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUUUG4(I) ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUUUG5(S) ACCCUUCCUU UCUUCGGGUG UC

14、CUUCCUCG CGCCCGCAGG ACCACCCCUC G-CCCCUUUG6(S) ACCCUUCCUU UCUUCGGGUG UCCUUCCUCG CGCCCGCAGG ACCACCCCUC GCCCCCUUUG CCR Path1(M) CGGUGUCGCU UCGGCUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC2(M) CGGUGUCGCU UCAGCUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC3(M) CGGUGUCGCU UCGGAUAUUA CCCGGUGGAA AC

15、AACUGAAG CUCCCGAGAA CCGCUUUUUC 4(I) CGGUGUCGCU UCGGCUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC 5(S) CGGUGUCGCU UCGGCUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC 6(S) CGGUGUCGCU UCGACUACUA CCCGGUGGAA ACAACUGAAG CUCCCGAGAA CCGCUUUUUC TL1(M) UCUAUCUUUC UUUGCUUCGG GGCGAGGGUG UUUAGCCCUU GGAAC

16、CGCAG UUGGUUCCU2(M) UCUAUCUUUC UUUGCUUCGG GGCGAGGGUG UUUAGCCCUU GGAACCGCAG UUGGUUCCU3(M) UCUAUCUUUC UUUGCUUCGG GGCGAGGGUG UUUAGCCCUU GGAACCGCAG UUGGUUCCU4(I) UCUAUCUUAC -UUGCUUCGG GGCGAGGGUG UUUAGCCCUU GGAACCGCAG UUGGUUCCU 5(S) UCUAUCUUUA UUUGCUUCGGC GGCGAGGGUG UUUAGCCCUU GGAACCGCAG UUGGUUCCU 6(S) U

17、CUAUCUU -A UGUGCUUCAG GGCGAGGGUG UUUAGCCCUU GGAACCGCAG UUGGUUCCU PSTVdPSTVd中国分离株与已报道序列在一级结构上的差异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 Comparison with mild strain from China in nucleotide sequence of the isolates of the mild, intermediate and severe strain of potato spindle tuber viroid(PSTVd)

18、PSTVd分离株系PSTVd isolates类病毒核酸长度Viroid-nucleotideChain length同中国弱株系相比核酸差异Differences from “the mild strain”of China in sequence变化位置Position of the changed nuoleotideMild:PSTVd-WA(未例出)PSTVd-F(未例出)PSTVd-India PSTVd-WB 359 nt358 nt359 nt 359 nt UAUCUGACACUGA 309(致病区)309(致病区)255(中央保守区)258(中央保守区)253(中央保守区)

19、PSTVd KF-6 mild type359 ntNoneNoneSevere:PSTVd-F-SL PSTVd S17-I-8 359 nt 359 nt AinsertionAdeletionUinsertionCdeletionGACAUdeletionC insertionUAAU deletionCAUGGA 48-49（致病区）122(可变区)140-141(可变区)232 (可变区)254(中央保守守区)310(致病区）311（致病区）320-321(左未端区)120(中央保守区)309（致病区）310（致病区）312（致病区）319(左未端区)Intermediate:PST

20、Vd-I-India 359 nt UAAUAUdeletion 120(中央保守区)309(致病区)311(致病区) 注：From Herold et al.(1992); From Owens et al.(1992); From Singh et al.(1993); From Gross et al.(1981); and From Gora-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

21、）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：8 g,1.6 g,0.32 g,64 ng, 13 ng,2.6 ng, 520 pg,104 pg最低检出率：13ng双向电泳检测双向电泳检测PSTVd PSTVd检测结果：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：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PSTVd434bp328bp 1 2 3 4 5资料检索、

22、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PSTVd pGEM-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 500 ng 50 ng 5 ng 500 pg 50 pg 5 pg 200 pg 50 pg 200 pg 50

23、pg NASH方法检测方法检测PSTVd专化性专化性测试（颜色反应）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 1、HaeHae单酶切含单酶切含PSTVdPSTVd单体的单体的pGEM-7ZpGEM-7Z质粒质粒 1 2 3 328bpHae/7Z空 Hae/7Z-PSTVd (bp) 正向插入 反向插入1 657 657 6572 458 458 561*3 434 434 4584328* 369* 4345289 320 * 2896267 289 2677174 267 1748142 174 1429102 142 125*1080 102 10211 80 80结果显示： pGEM-7Z重组质粒含有PSTVd单体，且为正向插入10 mM dNTP 2 l 引物(60 pM) 2 l模板(P7z-PSTVd单体) 2 lDEPC H2O 8