類病毒檢測技術(shù)培訓(xùn)

1、會計學(xué)1類病毒檢測技術(shù)培訓(xùn)類病毒檢測技術(shù)培訓(xùn)二、類病毒種類已知序列類病毒種類及其分類 The variants of viroid known sequence and their classification中名Chinese names英名English names縮寫Abbrevia-tion大小Length (nt)中央保守區(qū)Central conserved馬鈴薯紡錘塊莖類病毒Potato spindle tuberPSTVd356-360PSTVd柑桔裂皮類病毒Citrus exocortisCEVd370-375PSTVd菊花矮化類病毒Chrysanthemum stuntCSV

2、d354-356PSTVd番茄頂矮化類病毒Tomato apical stuntTASVd360-363PSTVd番茄雄性株類病毒Tomato planta machoTPMVd360PSTVd唇膏蔓薔薇隱性類病毒Columnea latentCLVd370-372PSTVd啤酒矮化類病毒Hop sturtHSVd297-303PSTVd椰子死亡類病毒Coconut cadang-cadangCCCVd246-247CCCVd椰子tinangaja類病毒Coconut tinangajaCTiVd254CCCVd啤酒花隱潛類病毒Hop lalentHLVd256CCCVd柑桔IV類病毒Citr

3、us IVCVdiV284CCCVd蘋果銹果類病毒Apple scar skinASSVd329-330ASSVd葡萄黃斑類病毒1Grapevine yellow speckle 1GYSCd1366-368ASSVd葡萄黃斑類病毒2Grapevine yellow speckle 2GYSCd2363ASSVd澳洲葡萄類病毒Australian grapevineAGVd369ASSVd柑桔硬葉類病毒Citrus bent leafCBLVd318ASSVd梨皰潰瘍類病毒Pear blister cankerPBCVd325ASSVd鱷梨白斑類病毒Avocado sunblotchASBVd

4、245-250ASBVd桃潛花葉類病毒Peach latent mosaicPLMVd336-337ASBVd錦學(xué)蘇類病毒Coleus blumei ICbVd1248CbVd1 類病毒結(jié)構(gòu)示意圖 The structure of viroid 馬鈴薯紡錘塊莖類病毒熱變性過程 Heat-denatured process diagram of PSTVd三、類病毒結(jié)構(gòu)馬鈴薯紡錘塊莖類病毒的五個功能區(qū) The five functional domains of PSTVd 馬鈴薯類病毒中國分離株核苷酸序列l(wèi) 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致病區(qū)的位置和結(jié)構(gòu)。圖解表示PSTVd-Intermediate的完整結(jié)構(gòu)，包括左未端區(qū)（TL,1-46/315-359），致病區(qū)（47-73/286-314），中央保守區(qū)（74-120/240-285），可變區(qū)（121-148/21

8、2-239）和右未端區(qū)（TR, 149-211）(Keese and symons, 1985),premelting(PM)區(qū)1-3（Steger等，1984）和二級發(fā)夾結(jié)構(gòu)1-111。下面為三個自然發(fā)生的PSTVd分離株系。水平箭頭指出PM1和VM區(qū)域（Schnlzer等，1985），“星號”表示中間株系和強(qiáng)、弱系序列差異。此圖引自O(shè)wens 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中國分離株與已報道序列在一級結(jié)構(gòu)上的差異中國分離株與已報道序列在一級結(jié)構(gòu)上的差異 PSTVd強(qiáng)、弱系及中間株系的核苷酸序列差異比較 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分離株系PSTVd isolates類病毒核酸長度Viroid-nucleotideChain length同中國弱株系相比核酸差異Differences from “the mild strain”of China in sequence變化位置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(致病區(qū))309(致病區(qū))255(中央保守區(qū))258(中央保守區(qū))253(中央保守區(qū))

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

20、Vd-I-India 359 nt UAAUAUdeletion 120(中央保守區(qū))309(致病區(qū))311(致病區(qū)) 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、類病毒鑒定1、指示植物接種鑒定 1）魯特格番茄 2）新莨菪2、電泳 1）垂直雙向丙烯酰胺電泳（2-D-PAGE） 2）往返丙烯酰胺電泳（R-PAGE）3、反轉(zhuǎn)錄PCR（RT-PCR）4、核酸斑點雜交（NASH

21、）往返丙烯酰胺電泳（R-PAGE）檢測流程圖核酸提取第一項電泳反向電泳固定銀染色顯色結(jié)果判讀PSTVd上樣量：8 g,1.6 g,0.32 g,64 ng, 13 ng,2.6 ng, 520 pg,104 pg最低檢出率：13ng雙向電泳檢測雙向電泳檢測PSTVd PSTVd檢測結(jié)果：檢測結(jié)果：樣品含有PSTVd，箭頭所指為類病毒核酸帶RT-PCR檢測類病毒流程引物設(shè)計擴(kuò)增產(chǎn)物分析（）以cDN為模板擴(kuò)增制備cDN第一鏈（反轉(zhuǎn)錄）樣品核酸提取檢測結(jié)果：檢測結(jié)果：PCR反應(yīng)退火溫度分別設(shè)定為55 和57 ，在57 下特異性好，可用來檢測PSTVd434bp328bp 1 2 3 4 5資料檢索、

22、收集、樣品采集類病毒病原鑒定:三種方法：（R-PAGE、2-D-PAGE、RT-PCR）含PSTVd pGEM-7Z質(zhì)粒利用HaeIII對重組質(zhì)粒進(jìn)行酶切驗證鑒定結(jié)果：含PSTVd設(shè)計引物、對含PSTVd的pGEM-7Z質(zhì)粒進(jìn)行PCR擴(kuò)增利用生物素biotin-11-dUTP代替dUTP制備探針進(jìn)行核酸斑點雜交反應(yīng)NBT/BCIP 化學(xué)顏色反應(yīng)Flu熒光素發(fā)光反應(yīng)試劑盒組裝生 產(chǎn) 應(yīng) 用核酸斑點雜交（）檢測技術(shù)流程探針制備終止反應(yīng)預(yù)雜交封閉孵育信號產(chǎn)生雜交漂洗點樣尼龍膜的處理 500 ng 50 ng 5 ng 500 pg 50 pg 5 pg 200 pg 50 pg 200 pg 50

23、pg NASH方法檢測方法檢測PSTVd專化性?；詼y試（顏色反應(yīng)）測試（顏色反應(yīng)）結(jié)果顯示：陽性樣品有斑點，陰性樣品不產(chǎn)生斑點。 反應(yīng)專化性強(qiáng)。核酸斑點雜交（）檢測反應(yīng)原理圖 1、HaeHae單酶切含單酶切含PSTVdPSTVd單體的單體的pGEM-7ZpGEM-7Z質(zhì)粒質(zhì)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結(jié)果顯示： pGEM-7Z重組質(zhì)粒含有PSTVd單體，且為正向插入10 mM dNTP 2 l 引物(60 pM) 2 l模板(P7z-PSTVd單體) 2 lDEPC H2O 8