基因分型技術(shù)簡介及應(yīng)用舉例

基因分型技術(shù)的應(yīng)用國家結(jié)核病參比實驗室中國疾病預(yù)防控制中心周楊2017-9-13基因分型方法簡介噬菌體型IS6110限制性片段長度多態(tài)性（RFLP,restrictedfragmentlengthpolymorphism)間隔寡核苷酸分型(Spoligotyping,Spaceroligonucleotidetyping)可變數(shù)目串聯(lián)重復(fù)序列多態(tài)性(MIRU-VNTR,MycobacterialInterspersedRepeatUnits-VariableNumberTandemRepeat)單核苷酸多態(tài)性(SNP,singlenucleotidepolymorphism)大片段多態(tài)性(LSP,largesequencepolymorphism)多位點序列分析(MLST,multilocussequencingtype)全基因組測序(WGS,wholegenomesequence)IS6110RFLPMunsiff,SonalSJournalofInfectiousDiseases2003，188（3）：356-363TomaszJagielskiBioMedResearchInternational2014,ArticleID645802,21pagesSpoligotyping直接重復(fù)區(qū)(DR)：36（35）bp的保守序列間隔區(qū)(spacer)：34-41bp的多態(tài)性序列，在不同菌株中順序保守，數(shù)目不同。在直接重復(fù)區(qū)設(shè)計引物，對間隔區(qū)進行擴增?，F(xiàn)在通用的spoligotyping方法中選用了43個間隔區(qū)序列。DRDRDRDRDRspacer1spacer4spacer3spacer2Supply,PhilipMolecularMicrobiology2000,36(3):762-771/applications/mycobacterial-interspersed-repetitive-units-miru-typingMiru-vntrIDMIRU4MIRU26MIRU40MIRU10MIRU16MIRU31VNTR42VNTR43ETRAVNTR47VNTR52VNTR53QUB-11bVNTR1955QUB26MIRU02MIRU23MIRU39MIRU20MIRU24MIRU27VNTR46VNTR48VNTR49CN032042733453444322411253213423MIRU20MIRU24MIRU27QUB26snpSreevatsan,SPNAS,1997,94(18):9869-74LSPTsolaki,AnthonyGJournalofclinicalmicrobiology,2005,43(7):3185-91Variablehost-pathogencompatibilityinMycobacteriumtuberculosis.Gagneux,Sebastien,2006,PNAS不同分型方法結(jié)果比較AnewevolutionaryscenariofortheMycobacteriumtuberculosiscomplex.mlst3R基因分型：修復(fù)(repair)，復(fù)制(replication)，重組(recombination)16s,16-23ITS,rpoB,hsp65PLoSONE6(1):e16020.doi:10.1371/journal.pone.0016020wgsCole,STNature，1998，393（6685）：537-544比較基因組研究（Comparisongenomeanalysis)基因組相關(guān)性分析(GWAS)應(yīng)用舉例疫情監(jiān)測療效監(jiān)測院感控制種群結(jié)構(gòu)系統(tǒng)進化中國耐藥結(jié)核病的傳播

Chongguangyang,clinicalinfectiousdisease,2015,61(2):219-227主要流行株的生物學特性

Zhou,YangpLOSoNE2017,12(3):e0174197全國設(shè)立了70個耐藥監(jiān)測點，納入所有涂陽病例2007年4-12月，共計納入并獲得3929例病例的藥敏結(jié)果：利福平，異煙肼，乙胺丁醇，鏈霉素，卡那霉素，氧氟沙星基因分型：spoligotyping,24MIRU-VNTR,WGS幾個常用的參數(shù)和分析方法

/wiki/Minimum_spanning_tree種群結(jié)構(gòu):

共分為7個主要克隆復(fù)合群(clonalcomplex,>10），屬于4個世系(lineage)主要流行株—高成簇率:北京基因型和歐美型-亞型3最流行的基因型:5個最大的簇基因型總例數(shù)成簇病例數(shù)成簇率%95%CI北京基因型2208106148%46.0-50.2歐美型-亞型13456519%14.9-23.4歐美型-亞型22252310%6.6-14.9歐美型-亞型31163328%20.5-37.6其他基因型239219%5.5-13.1MST和成簇率VNTR基因型（≥20）24VNTR*病例數(shù)譜系VT007273335444432658253213423133Beijing(133)VT02827333544443255825321342383Beijing(82),Manu2(1)VT11127323544443265825321342335Beijing(34),T1(1)VT06427333544443245825321342332Beijing(32)VT04827333544443265725321342325Beijing(25)抗結(jié)核藥

北京基因型(n=2208)歐美型-亞型1(n=345)歐美型-亞型2(n=225)歐美型-亞型3(n=116)PValueResistantstrains(%,95%CI)

全敏感1389(62.9%,60.9-64.9)

221(64.1%,58.8-69.1)

131(58.2%,51.5-64.7)

56(48.3,38.9-57.8)0.006任意耐藥

INH476(21.6%,19.9-23.3)61(17.7%,13.8-22.1)38(16.9%,12.2-22.4)18(15.5%,9.5-23.4)0.08SM602(27.3%,25.4-29.2)85(24.6%,20.2-29.5)75(33.3%,27.2-39.9)51(44.0%,34.8-53.5)<0.001EMB195(8.8%,7.7-10.1)18(5.2%,3.1-8.1)11(4.9%,2.5-8.6)4(3.4%,0.9-8.6)0.007RFP304(13.8%,12.4-15.3)32(9.3%,6.4-12.8)13(5.8%,3.1-9.7)8(6.9%,3.0-13.1)<0.001KM45(2.0%,1.5-2.7)6(1.7%,0.6-3.7)2(0.9%,0.1-3.2)5(4.3%,1.4-9.8)0.195OFLX107(4.8%,4.0-5.8)9(2.6%,1.2-4.9)6(2.7%,1.0-5.7)0(0.0%,0.0-3.1)0.013MDR266(12.0%,10.7-13.5)31(9.0%,6.2-12.5)10(4.4%,2.2-8.0)7(6.0%,2.5-12.0)<0.001XDR19(0.9%,0.5-1.3)1(0.3%,0.0-1.6)1(0.4%,0.0-2.5)0(0.0%,0.0-3.1)0.464多項研究顯示，北京基因型與耐多藥結(jié)核病顯著相關(guān)?；蛐捅本┗蛐?n=2208)歐美型-亞型1(n=345)歐美型-亞型2(n=225)歐美行-亞型3(n=116)P-valueNumberofstrains(%,95%CI)sus1510(68.4%,68.1-68.7)243(70.4%,69.9-71.1)145(64.4%,64.0-65.2)61(52.6%,51.7-53.4)0.411S394(17.8%,17.6-18.2)70(20.3%,19.7-20.9)67(29.8%,29.3-30.5)47(40.5%,39.7-41.4)<0.001R96(4.3%,4.1-4.7)17(4.9%,4.3-5.6)5(2.2%,1.8-3.0)4(3.4%,2.6-4.3)0.008SR208(9.4%,9.1-9.8)15(4.3%,3.8-5.0)8(3.6%,3.1-4.3)4(3.4%,2.6-4.3)<0.001據(jù)BuuTranN（PLosOne,2012,7(8):e42323)報道，北京基因型鏈霉素耐藥株顯示出比敏感株更高的成簇率。爆發(fā)疫情處置

王勝芬，中國防癆雜志，2009，31（10）：573-578某校3個月的時間內(nèi)發(fā)現(xiàn)67例結(jié)核癥狀病例培養(yǎng)方法：L-J改良固體培養(yǎng)基生化實驗：D-甘露醇，肌醇，D-山梨糖醇藥敏實驗：異煙肼，利福平，鏈霉素，乙胺丁醇，卡那霉素，卷曲霉素，丙硫異煙胺，對氨基水楊酸，氧氟沙星，左氧氟沙星，利福噴汀，丁胺卡那霉素鑒定及分型：PNB-TCH，hupB，dnaA-dnaNIS6110，NTF-1IS6110，16s，16-23ITS，hsp65，spoligotyping，MIRU-VNTR，結(jié)果1株慢生長，2株快生長，均為非產(chǎn)色結(jié)論：慢生長菌為結(jié)核分枝桿菌，北京基因型現(xiàn)代株；快生長菌未豬分枝桿菌慢生長菌快生長菌D-甘露醇-+肌醇-+D-山梨糖醇--PNB-TCHMTBC?INHSRSMSREMBSRRFPSRKMSRCMSRAMSSPTASRPASSROFLXS?hupB645/318bp\dnaA-dnaN2000bp\NTF523/223bp\SpoligotypingSIT1\MIRU23325173533\16s\偶發(fā)？豬16-23ITS\偶發(fā)？豬hsp65\偶發(fā)？豬限制性內(nèi)切酶\豬北京基因型的起源進化

Merker,MatthiasNatureGene