GB/T 43166-2023 燕麥?zhǔn)人峋鞴蟻喎N溯源檢測(cè)方法（正式版）

ICSCCS65.020.20燕麥?zhǔn)人峋鞴蟻喎N溯源檢測(cè)方法國(guó)家標(biāo)準(zhǔn)化管理委員會(huì)IGB/T43166—2023本文件按照GB/T1.1—2020《標(biāo)準(zhǔn)化工作導(dǎo)則第1部分：標(biāo)準(zhǔn)化文件的結(jié)構(gòu)和起草規(guī)則》的規(guī)定起草。請(qǐng)注意本文件的某些內(nèi)容可能涉及專利。本文件的發(fā)布機(jī)構(gòu)不承擔(dān)識(shí)別專利的責(zé)任。本文件由全國(guó)植物檢疫標(biāo)準(zhǔn)化技術(shù)委員會(huì)(SAC/TC271)提出并歸口。1GB/T43166—2023燕麥?zhǔn)人峋鞴蟻喎N溯源檢測(cè)方法本文件描述了應(yīng)用全基因組單核苷酸多態(tài)性(wgSNP)方法對(duì)燕麥?zhǔn)人峋鞴蟻喎N進(jìn)行溯源檢測(cè)本文件適用于燕麥?zhǔn)人峋鞴蟻喎N的分子溯源檢測(cè)。2規(guī)范性引用文件本文件沒(méi)有規(guī)范性引用文件。3.13.33.4具有共同祖先的各物種間進(jìn)化關(guān)系的樹(shù)。注：在進(jìn)化樹(shù)中，每個(gè)分支節(jié)點(diǎn)代表其各分支的最近共同祖先，而節(jié)點(diǎn)間的線段長(zhǎng)度對(duì)應(yīng)演化距離。單核苷酸多態(tài)性singlenucleotidepolymorphism;SNP在基因組水平上由單個(gè)核苷酸的變異所引起的DNA序列多態(tài)性。在全基因組水平上由單個(gè)核苷酸的變異所引起的DNA序列多態(tài)性。4病菌的基本信息Pseudomonaspseudoalcaligenessubsp.citrulliSchaadetal.,1978單胞菌科(Comamonadaceae),噬酸菌屬(Acidovorax),西瓜亞種(Acidovoraxavenaesubsp.citrulli)。該病菌的其他相關(guān)資料見(jiàn)附錄A。2GB/T43166—20235方法原理燕麥?zhǔn)人峋鞴蟻喎N溯源檢測(cè)方法的原理是通過(guò)全基因組測(cè)序技術(shù)發(fā)掘物種的wgSNP位點(diǎn)，基于得到的單核苷酸多態(tài)性信息構(gòu)建系統(tǒng)發(fā)育樹(shù)，進(jìn)行聚類分析，從而對(duì)物種進(jìn)行溯源歸類。6儀器和器具種針。7試劑和培養(yǎng)基溶解后，每份10mL分裝到玻璃試管中。以上培養(yǎng)基均在高壓滅菌鍋中121℃高壓滅菌20min,備用。8溯源檢測(cè)方法8.1檢測(cè)流程對(duì)燕麥?zhǔn)人峋鞴蟻喎N進(jìn)行溯源的流程包括菌株的活化、DNA的提取與純化、全基因組測(cè)序、數(shù)據(jù)8.2菌株活化在超凈工作臺(tái)環(huán)境中，將保存的菌株用LB固體培養(yǎng)基用接種針取菌采用平板培養(yǎng)皿上劃線法活化，于28℃恒溫培養(yǎng)箱中倒置培養(yǎng)2d～3d備用。8.3DNA提取與純化使用CTAB法或商業(yè)DNA提取試劑盒，對(duì)基因組DNA的提取方法見(jiàn)附錄C,提取后通過(guò)PCR擴(kuò)增儀對(duì)其進(jìn)行PCR擴(kuò)增，并用電泳儀對(duì)PCR產(chǎn)物進(jìn)行電泳，最后使用凝膠成像系統(tǒng)進(jìn)行檢測(cè)。用核酸蛋白檢測(cè)儀測(cè)定DNA濃度及純度后，保存于—20℃冰箱備用。8.4全基因組測(cè)序以模式菌株(REF)或來(lái)源明確的菌株的全基因組序列為參考基因組，采用全基因組鳥(niǎo)槍法(WGS),利用測(cè)序儀對(duì)構(gòu)建的PE(Paired-End)文庫(kù)進(jìn)行2×150bp測(cè)序，測(cè)序有效深度(SequencingDepth)應(yīng)達(dá)到200×,即測(cè)序所得到的堿基(bp)總量與待測(cè)物種基因組(Genome)大小的比值應(yīng)達(dá)到200。8.5數(shù)據(jù)評(píng)估質(zhì)控對(duì)待測(cè)物種測(cè)序的原始數(shù)據(jù)(rawreads)通過(guò)通用生物信息分析軟件進(jìn)行質(zhì)量評(píng)估。然后通過(guò)通3GB/T43166—20238.6序列比對(duì)按照通用生物信息分析軟件列出的流程開(kāi)展序列比對(duì)：a)使用通用生物信息分析軟件將待測(cè)物種基因組有效數(shù)據(jù)與參考基因組(8.4)進(jìn)行比對(duì)；b)使用通用生物信息分析軟件轉(zhuǎn)換比對(duì)結(jié)果的格式，并對(duì)其排序；c)統(tǒng)計(jì)比對(duì)結(jié)果；d)使用通用生物信息分析軟件進(jìn)行重復(fù)序列的標(biāo)注(MarkDuplicates);e)根據(jù)比對(duì)結(jié)果進(jìn)行冗余序列(duplicatereads)分析、插入片段長(zhǎng)度(insertsize)的分布等分析；f)利用通用生物信息分析軟件進(jìn)行深度覆蓋率統(tǒng)計(jì)分析。8.7SNP檢測(cè)利用通用生物信息分析軟件的識(shí)別單倍型(HaplotypeCaller)算法分析待測(cè)物種基因組和參考基因組之間的基因型差異，對(duì)待測(cè)物種的SNP進(jìn)行分析，得到變異信息并統(tǒng)計(jì)變異位點(diǎn)。將待測(cè)物種的全基因序列單核苷酸多態(tài)性位點(diǎn)按相同順序串聯(lián)起來(lái)，得到一條fasta序列(參考附錄D),然后將每個(gè)菌株等長(zhǎng)的fasta序列輸入到建樹(shù)分析軟件，再通過(guò)系統(tǒng)發(fā)育樹(shù)鄰接法(NJ法)基于wgSNP構(gòu)建系統(tǒng)發(fā)育樹(shù)，對(duì)菌株(含參考菌株)進(jìn)行親緣關(guān)系分析。9結(jié)果判定根據(jù)待測(cè)物種序列在系統(tǒng)進(jìn)化樹(shù)上與已知來(lái)源的燕麥?zhǔn)人峋鞴蟻喎N的分支聚集結(jié)果進(jìn)行判定：———如果待溯源物種與已知來(lái)源物種序列聚為一支，支持率為100%,即可判定待溯源物種與已知溯源物種具有相同的來(lái)源地；———如果待溯源物種與已知來(lái)源物種序列聚為一支，支持率低于100%,即可判定該物種與已知溯源物種疑似為相同的來(lái)源地；———如果待溯源物種未與已知來(lái)源物種序列聚為一支，則判定該物種與已知溯源物種不是同一來(lái)源地。10樣品與原始數(shù)據(jù)保存分離得到的菌種轉(zhuǎn)移至LB液體培養(yǎng)基，在恒溫?fù)u床中28℃培養(yǎng)36h,加入20%滅菌甘油，置于-80℃條件下保存至少12個(gè)月或采用無(wú)菌水保存法，嚴(yán)格防止擴(kuò)散，保存期滿后，經(jīng)高溫高壓滅菌處理。10.2結(jié)果記錄保存和審核人員簽字。PCR產(chǎn)物凝膠電泳檢測(cè)需有電泳結(jié)果照片，測(cè)序全基因組序列需要保存電子文件。4GB/T43166—2023(資料性)A.1分布var.saccharinus)、黃瓜(C.sativus)、南瓜(C.moschata)、西葫蘆(C.pepo)、番茄(LycopersiconEsculen-A.3為害癥狀5GB/T43166—2023(資料性)燕麥?zhǔn)人峋鞴蟻喎N溯源檢測(cè)流程燕麥?zhǔn)人峋鞴蟻喎N溯源檢測(cè)流程見(jiàn)圖B.1?；罨闐NA提取與純化全基因組測(cè)序數(shù)據(jù)評(píng)估質(zhì)控序列比對(duì)結(jié)果判定樣品與原始數(shù)據(jù)及保存圖B.1燕麥?zhǔn)人峋鞴蟻喎N溯源檢測(cè)流程6(資料性)基因組DNA制備C.1DNA制備使用下列CTAB方法提取病菌DNA,或使用商業(yè)化試劑盒提取DNA。a)菌株的培養(yǎng)及DNA制備的前處理。1)將菌株接種于裝有LB液體培養(yǎng)基試管中，置于恒溫?fù)u床上28℃搖培28h。2)在試管中加入0.1mol/L磷酸鈉緩沖液10mL洗下菌體。3)將細(xì)菌懸浮液轉(zhuǎn)移至滅菌的離心管中，置于冷凍離心機(jī)中12000g下離心15min,棄上清液。b)DNA的提取。37℃置水浴鍋中水浴1h。2)加入等體積的三氯甲烷/異戊醇(24:1),混勻，10000g離心5min,將上清液轉(zhuǎn)移至新的滅菌離心管中。3)加入等體積的酚/三氯甲烷/異戊醇(25:24:1)混勻，10000g離心5min,將上清液轉(zhuǎn)移至新的滅菌離心管中。4)加入0.6倍體積的異丙醇，輕輕混合至DNA沉淀下來(lái)，10000g離心5min,棄上清液。C.2DNA濃度及純度測(cè)定用核酸蛋白分析儀測(cè)定DNA的純度與濃度，分別取得260nm和280nm處的吸收值，按照公式(C.1)計(jì)算DNA純度，按照公式(C.2)計(jì)算DNA濃度：p=50×OD?60…………(C.2)p——DNA質(zhì)量濃度，單位為微克每毫升(μg/mL);OD?260nm處吸光光度值，核酸的吸光度；OD?80——280nm處吸光光度值，蛋白質(zhì)的吸光度。PCR級(jí)DNA溶液的OD?60/OD?s?比值為1.7～1.9。(資料性)參考菌株wgSNP位點(diǎn)序列參考菌株wgSNP位點(diǎn)序列>REFCTTTTAGTGATTCAACTGGGACCGTCAGGCCTCGCCGATCGTTCCGGTGGTGAAACTTCATGCGTGGGTATCCGGCTCTATCGTACTCCACTCGACTTAGTCGGGTCGGGCCAGGCCTCACGTTCCTTAGCGATGTTGAATCTCCACGTGCTGGGTGTTTCGCGGGCCCGCGGCATGGTCTGTGCTCTAACGGCGTCACGACAGCCTTGTCTGACTGGGATCAGGGGTGCCGGTCTCGGACCTACTCACGTGGCCGTCGGCCACAITCGACGCACGGTGGGTATCCGTCCCAGTAGCCTATGCACGCACTTTGTGCCGTAGGGCGGGGCGGTGTTGCGGGCGGAGGGCGCIGGACGGGTGGCACGCGGGTGCATACTTCGCIATGGTAAGCTGATGTTACACCGTIGCCTGTAGGCAAGGGTGCCGGAGTCTGCCGGACGAGCGCCCGGGGTTTCTGTTATAATCAGCGGCGATCGCCCTGCCGTCGCACTGGAGTGCTAGTAGGGTCTGCCTACATAGGGAATTTTGAACGCGGATTCGGACGTGACCAGCTGGTCTCCCATACCGCTCGTAAAACGGCGGCTGAGCCCGTGGCCGTCCTCCGCGCGATCCGGCCCGTCGCCTCGTCGTATCCGGCCTTATGGGGAGGGATCCCCGCCTCTCCAAGGCGCCAAGTGGTCACCACTGTCCCTGCTCGGTCCCCCCTCATCGGCCGGGCCIGCITGCGCCCTItTCCGCAI'"TGGGATAGCGGGTGAGCITTCCTTCCGTGCGGCGCTTGCACAGGAAGGGGTGGAGTCTTGTTATCGGACTAGTGAATAGTGCCCGATCCTGGATCGATTGCACCAGCTTATGAGCTTGGGATCGTGAGAGGCGAGGACGGTCCACCCTCAGGGCCGCAAGTTTGTATATGTCTGGGAGCTCACGCAGCGCTACGAACGGCGGTAAGTAAGGAGGGGCACCCTGACCTGAGTGTTCGTGCCGGATGCCCACTACCCTTTCCACCAGCTTCCCCCCAGATGTGGCAGAGACTCTGTTGCTTTACTGTGTTrTCCTAAGGAAGTCATCGGGAATAACGAGATGAGACCATGTCTCGTCCACGATCGGCCGCACGCTCCCCAGTGCGACGGGTGCCGCGTGGGAGCTGCGAGAACAAGGCGTTCTGGTAAAGACCGACTAGCTGGCCGGCGTTCTGGCGCGCCGGCTCCGACACAGCCGGTCGCACGCTGATCGACAACGTTGCCTATTTTCCAGCCTGCCTGACACCAGGCAGGATTGCACTTTGGGTGCGGCGCATCTGGCCCCCTCCCCTGATCTCGATGATCATACCAGCTTTGCACATCGCACGCGTAAAGGCTACCGCTGTGGCCAGCCCCAGATCGAAGCGCCGGTGTACGCTATCAAGGGCGTTTICACATGTCTGCCAGAGGTGCTCGGCTGGCGGCCCGACTACATAGCCAACGTCGACTCAGACATTGGCAGTGCCTCTCGGCGCTGCTTGTCGTTCAATGGCTCGCCCGCTATTGGGACAACCTGCGGTCAGTTGTTCTAGCGGCTGCAAGTCGGACACCAGTGTGCGGTCGCGCCTCGTCGCCTGACACCGATGTCACGCGTTTTCCCGGAGGCAACGTGTGGTGTGTGCGTAATGCTCCTCGGGAATGGAAGTGGGGGCTGCGGACCGCCCCGCACTAGACGTGACCGGCTCTGTGTGCGACCTTGTAGTGAGGTGCACCCAGTGAGTTGGCTGGGTCTCTGTGTGGGAGCTGCTTTAAACGTGTAGTGCGTACACGCCAGATCCATCAAACCCCCAGATGCTGTGCCGCTCGGCGTCTATGGTGAGCCAGGGACGCCGGGCGTGGGCCGCTCCGGTGCGAGTCTGGCTCCGCGCTTCTCGGTATGCGCCTCCTGAACGGCTGGGCCCGACGATCGAACACCGACGCGGAGTGTTGTGTGCACCCTAGCCCGGCCGTAGTGCCTCGAGCCTTGAAGGCGCCTTGGTTAAGGCACAGCTCTGAGCCCTGTCCTGTTTCTGTGTGCGTCCGGCCACGATCCGAAACACCGGGGCGCGTGATCCGCCGGGGTCCTTCGCGCCGCGCGTCCTATCAGCATCACTTGGACGCGGAACGTGCATCGCTCACACTGATTCACAGGTTGCCGCACTGGAGCCCCCGACGACCGCCGGGCAAGAGGTCAGCATAATCGCCCAGGACGGCCCGTCTGCTATCGGCGGCTGTGGTGTTGTTCTTTTTCCGB/T43166—2023TTGCCGCCCTCGGAGCACTCCCGAGGCATGGCAAGATGTAGTACTCTCGAAATAGCAGCTCTACTACGATTACGACGGTTTCTCCCcCCGTGGAGCGGAGGCAAGCGAGGTTCTGGACGGCTGCTCTTCGGAGGCGCCCCGGGTTACTCCGTCGTGAGGCGTAGACAGCCACTATGGCTTATCGACAACTTCCTTAATTTTCCTCCATAGCCGTCCGAAGTTCTAATACAACTGCTATAAATCCCAACCTACCGGGTACACATGTTGGTCGTGTTGTAACTTAGGCGATAGTTCGTTTGATGTAGTATTCACACTTTTCGTCAACCCCGACGGGCCCGCGTGGGCGACCGGGGGCGCCTCGGCGCCGCCACAGAAGCGTCCGGGGGGTCCCGGGCTTGCTTAAGCCATACGGGGTGTAGTTCTCCCTACGAAGCTACAGGCTTTTCCAGCCACTGTTGCATAGTTACCAGATTAAGCCTTCACAAACGCCGTGCTCGCGACGCGGGCCACTGCGTCGGACTATCACTACCTTGTTATATCCGAGACGCAACGAAGCCCGCACGCCGTTAGGCAATGGTTTTCCGCAACGCCCCGTAAGGCCGTACCATAGTCCGCGCCTTCCGCCGCCGCATCAGAGAGGGCGATCCGCCTGCGAGGTGAGATTGGGGGGCAGGGCGATGCCCCGGGTGCGTACTTGGCACCGCAGATACTTCACATTTGTTGTGCGGCAGCCCTTAGTCACCAGGTTGTCGACGCGGTCATGAAGCCGTGCGCCACCTGGGCCCACCCGCACGCCGGTCACATGCGCGAAGGTGGTTGCCGAGCGCCCTTGGCGGGCAACATACTTCCGCCCTCCCGGCTCGTGCGTGTCGCCCCTCAAGACGCACTCACAAGAGAGTCCGCCCCTAATCTTCCCTGTCAITATACCCCTATGGTCACCCCTAGCAGCGTATTGAACAGTGGTCACCGAGGAGGCCGCGTGTCCGGCGCGGACCCCGCTATACTTTCCACCTCCTTTGGTTGGGATGGATGGCCTTTATACATTCGCGGTTACAACATCGTTAGGTGCGTCGTCACCGTATCCTGGCGAGCTCACCGGCTTGGCCACTCTCGGTCGGCTACTCAAATTAATCACCGCCCGACCACGACTCTCTGGGCCAACCCGGCTGTGGGCTTTGGTCATAAACATCGAGGTGCACACTAAGGCTATAGGACCGCAATTGAGGCAATCAGTCTGACTTACCGCTCTCGACCGTAGCCATCAITTACCCTGGGTACTTITGTCATGTGAACCCCCCGTCCTCCACCCGGAGGATTTGGCGGAACTGTCACACCCTATTCACACTCGGCTCCGCCGCGGGCAATCCCAGTGAGAGCGCGGCCTGCATCCCCTTGGCAATTCAAATCTGTGAGCTCATCCCATCACAGCCGACGGATGGTGCCCTTTTGCTGTTTGCACGGCCTGTCACCTGGCGTCATTTCCATCACATCTAGTACTCCGCCAGGGCGGATAAGAGCGAACTCCTTAAGAACTGCCCCCGGGGCTCATCCCCAGGTGGCAGGTCCTATAGTGGCTCAGCGGGATATAGCAGGAGCCACTGCTAGATAAAGITGTTGAGGCTCTGACGCTTACCGCCGGCGCGGTGTGGTGTCGACGCGGCGCTTGTGCTCCCCGGTGCCCTCTCGTCGCGTCCCCGCCGTTCGGCAATGGGAGGCACGGGAGCGCTGGTCCGCCCAGTTCGGGAGTTGCGTTTGCCGGGGGATGATACGGCCACCACGGACCCATTTGGCTAATATGAGCGGCCAAAGTGTCTTCGTCCTATGCCTGCCTGGTGGTGCGATGTACCGACGCTTCCTGCACTTGACTGAGATATGCAGcCGTTAGCCCGCGTGGCCCACGTCCITrICACTGTAICTGCCGAATCGCGTCATCTACGACAGTAATTGICGACGAAGTGGGACAGTACATATAGCCCTTTTACAGGAGAGTCCAATGACTACACTCTTCCCAGGACCGGAGATGGCTTCTTGTGGGGCCTCCTACCCCTGGCCCCCTCACACCGCGCCGAAGTTTGGTTAGATTGATCTAAGGCCTCGCCCCACGCCGCGCTGGTCGAGTTGAGGGACCCATCCCGTCGGTCGGCAACATGAGACATTCGACGTTCTGCCTTCATGGCTACAAAGGCGGTTCTTCAGTCAATCCAACCTTCGCCCAGGACCCAGGTGCAATAAGTAAGCTGGCGGTGAAGCCGCCCACGTGCCCGGATCCGCCGCCCGGCGGTCCCAGCACGTAATTGAGGAGACGTGTCCCGCGAGCGTGATCGGTCTGGCCTACCTCGGAGCCCTCCTTCGTATCACAATGCAAGTAACTGCTCGACTTGTGCCCGAGCTCAGGTATTCGCGCACCCCCACGGGAGTACAAGGCGAAGGCTGCCCTTACCTCGAGAGCGCTGATTTGCACCGTGCGGTGGCGACAGTCTTTGTTAGTAGGGGCGCCAACATCGGCACCTACTGGTCATGTTTCCACCGCGCGAGAACACAGTGCGGGTTATCACTGGTTCCGCGCTAACGCCGCTAACAITCICCGCAATGACCCGTTGGCTTIAAAACGCGCGAACAAGGTGGCACGTGTAACAGGTAATACGTGATTGCCGATCTTCCGCGCGTTGCCATGGCGAGAGCGGGCCCATTGGCATGATTTCCCCGGTCCGCCTGGGAGGGGCGGCTGGGCGCCGGGCGTTCCCGCTCCCAGGCTACACCGGACGCCAAACGACATTCTGCGCGCCGCCGTCCACCCCATTGCTTACTCCGTCCGAGCGCCAGGGCCGCGAGGCATGCGGAGCTCAGGTAGATATCAACTCTCAGGGGCTCCCCGCGGGOIVO9IIVD00IOVDVVVD999VDvVO99VIVOp3Vo0D9090I9VVIIDIIDVII009VL?DVODI9VVDloVD100300vov000DL3oVviv391ovvv?p1V3Lo199ovVDDIVlIVO139D00VVlLD91VDLVO000VvoVODLVLIVDo0VOOVDo0VDLLDL9D00vov30000LVD0V000V90VVi3000L00V!009000VO.IVDLVODL?D30vVo9V39VO00Vv?V0p0VVODIVOLLDVOD30V9000D00LOV300VOVID9000IVODVIOVVOD00VOVO99IVDV90090099ID909090D0LID00D00IV90D909VD0DV9DIOIIVDOVDo0VODIIDIOVD99LI90009V9D9009VD9VDDDI9VDIVD009LDIVOOI0IDLDO1vOVDIVDD130VlIVDLDVOl3VD9DVD3913091VD3VDVID1VLD101313090V9130131D30D001001V300L?IVVLLlL3000000VDivVOVi5L000009VivVOD0090VO?!)L1090V9!)L1001D0VDpDVDo.IV?vvovDvvVpoVD0D09VVDLDIDLLDIVD00LDDVVVIDVOvvVDvovVoovVVD9V39VD0V9990VIDV9VDoVV99IVODLIDI09DIDLID9VD0DVIO90VOID00V999090VOVVIII9D999V9009I39V9999VD9ID9IOVDI9OVIVD3VOI99ID99VVD9VID39D9V9909VDDpDVL?vVVD9DVVD139VVOD39D1910D00300D9D0990vvop0VooVopDVDVDDLD90Vip0V5lDV1501vVVooVVIDIVoovvvVO0010VODVV3oovvOV1povvo1vovovvVIVVooV3l00Ll)13VDD?00VLLLDVLIIVDvvvVvovvpVL?VVD0D0vVVLOVIVVIVOVDLDD1V300IVVLVOL00VVO09VIVVIDLIS0IDOV9909VOVVIVOvVp0VVIIDI909999VID0VVVI90DIDDI3D09090VviDDLLl19DVVLLL33L3lIVDD09lLDL3DIVODL3313DlLlDViolIVOl1009150ll91903020VIVOD00llDOVLpV301300v3o5V15ovVO00050LLl5V50000010LDo0VO0IVDDLlDVpoV!ILD00vvV30p01D00v?V0900005DvDVD00030pVDD9000DL?D90DLID00200001DVDoVpVOIVIVODDLDD2IDVOVD50VOOVOVID?IDID02DOVDIDOVVOOIODVOD0VVIDI3D00IOVO9DI900ID0II9DV999999LLDDVvOVo0V3090VDL9009LLISLIOVLI5DVVI90VvOVOVLD9VOL9D1909VDD991oVVIVIDDL1D0010190130VvD9230V0o9v?V33900v30009vOVDL3000VIDVVDDLIDLID09V9D009LO99LIDOILIVIVLDDIDDVID9992IDVLIDOLIDVO9IDLIIDDVVDSV9VV?vVVID9I099IVVOIVVIVI900VLD99ID0D09DIVOVIVO90VID99D99V999VO09VDD0219D00D1vDD0DD9DllLll9D1DD9D9V3sVDVOD00D90DD0D1VVLD0D00v3OVLlovpovvDLLDD00L09DLIDLD90000001D9L9DVVpoVD01D0DLDV39IVIOVD001D01501D90VD001D9V3o0VO990I9OI09I9VO90090990D932LD9VD9090099093990VO9D?IDI90VIDVOID01DVL5000I300900LD0LLDLD000001500000VODLDOVIVOVOD0V?vVIVO0D?ID909V000VI9999VIOIO0V9D90I99009VO99LD99IOVOLIVO9I9099IVOOIDI0VVVOVDID00VID900I9OVIVD9V3OIVIVIVOVVD9IVOIDV9DI90ID9VO99IOIIVVIDVIVD90ID9VVD9I9VDOLIVD0VDD00D0019VovvIVDl130IvVDVDDOIV139DVIDLLll9VVllVVlLD13VL?vVV13009VOLIVL10VoovDVDVIVODLDDL5001LpVVlIVLLIV3o1VO0900vovooV5090V5000LD00000001ovVOLDD0D0IVD000000voVD30LD000000330V.LLDDLDD00001D0VVOD.L?D?1000VDoVD0VODVLID90VoDVVDOID9IDOVOOI3099VOvDo0VOIVOVI50VD99990IVOVD999VIVV99DL3DV9509I99900V?VVIVVO99VVODDIVO90IDIVOIDIVIVVDSVDODV990990909I9D0VD9090V3D00D9VDoVVDL3D01llL39VD019DDD31D9130DD13309DVL3D0D9990030D3L3VLDVlL000D0000VIvOVIVLD000D0EVID0VID01vVVo010Voov?v0000001901o3VOLDLD0GB/T43166—2023GTCGGCAGCGCGGGCGGTACTGGGTAGCTGCAACGCTCGGTCATACCGGTAGATGAGCACCCAAAGCATCATAAGAACGTAGACAAATTCTGTTTCTCCTCAGAATGGATGCTTAACCTATAACCGTACCTTTTTAGATCCTGGAGCCTTTTAGTCGGGGGGGGCGTTACAATGGGGCCAGGTTGGGAGTAGGGCGGCAGAGCCGCGGGGCGCGGAGAGTCGACGGCGCTGTCCATTGTCATTAAATACTAACTAGTTCCGGCCAGTCGGGACCAAGGGGACACCGCGGACAGAGGCAGGACTGAACGGATCGCCGAGCAGTAGCATTAGCACCCTTGGGCTCGGTCGAAGAGTAATGTTCTAAACACATCCCTCCGTCCAAGGTATTTGCGTCGGCGGCCCACGCGAGGTCTATTTGCGGCTATCTGTTCCGCACCCAGCCTCCTAAGTAGTCCTAGTGACACCTCCCGGACCCGAATCAACTCGGTAGGCAAGTGAGTTGGCTGGCGTAGATACCCTGGATTATAGGCCGTGGAGGGATGCGATGTCGTTTATGCCCCCCAAGGATCTCGGCACGTGGGATCGGAAGAGGTCGCATCCCTCGCTGGACCCGCTTAAGCGGACTTAAGGGGTGGGTCCACTGCCGTCCTCGCGTCATGTGCCGGCTGGTCCGTGTGACTCACCGCCACCCCCGTCGACCAGCTGATGGGTCGCGTTATTGTGGCCGCAGCCCACTATCATGGGCGATGACACCACAGGCGGCGTAAGTGCGTGTTGCAAAGCGCGTCATTGGACCTTTTCTCCTACACCTTAGTGTCGTCGAATACGGGGCTTCTCACCGTGTAGGCCTCCGACGGGAACCTTCGTCGCCTCCGGGTTTGCGACTCATGGTCGCGTCCGGAAGCGCCTCATAACTTGTACCTATACAACACTACACCGCCTCCGCCTATCCTCTTGTTTAGAAAGCCAAGTCATCGTACCGCGCCTCACCGCATCCATGGCGGATAGCCTGTCAAGATTGGCTACGCGGGAACAAGGTTGTGTCCCATCCTAGGTTGTAGCTCGGACGGCTCTTGAGATCGCCGTTGCACTGGACGCGGGCCGTCGTCGCCGCTAGCGTCCAGTGATCCAGCCGCCGGGCGAAGCAGGTAGGTCTCGGCCACACGCACATCCTCGCCGAGCGTACGGCCCCGAGAGGCCAGTAGCGTGCCGGTCTCATGGAACAACGTGCCTCCGCACGGCGTCACCGGTCTGCTAGTCAGTACGAATCCCGTCACGATTTGACTCCTCGGGCCGTACGTACCGCCAGGACGTGGGATTCACGGGGCGAGGGAGTAGGGGACGCACGAAGAGTCAGCCTCTGGCGTTGGGGGCGGTAGAGGTAGACGACCATCAGTGGCTGGCCATCGCATGATTGCCGGTCGCGCACCACGTCCGGCAGCCGGGTACCCCGCGACCCGGGACGGTCAGACTCGGGCTCCGAGAGAACACGTTTACGCGGCAGCCCTCCGTCGAGTTACTCACCCCCGCGGGTGCGTCGACGGGGCGGAGCGAGCGTGACGTGCCGGTGCGTCCGCGTCCATCTGGTCTTCACAGGGGTTGGAGATTGGTATCGTACCCACGAACTCGAACAAGGCGTAAATATTTCTAGAAAACTCCTAGCAGAGTTCGAAAAAATACGCCCCTAGCGCGATCCCGCCCAAAAGCGGTGAAACACGCTCCTGCTAGTAGCCCACTTCTTAATCGTATTGAATCTACACTGACAGTGACCACGAGAAAGTTCACTGACCGAGCTCGAGTCGCCAGCGCCTTCTTGGACTAGGGGCGATTGTAAAGTTCCCTAAAAACCCAAAACGCCGCGCGTGGTGTCGACCGGGAATCGTCGGGATACGTGCGTTTTGTGGCACATGACGGATCCCTTAGGCCCATGACCTTGCGAAGCTGTTCACCACGAAGTATGGGCGCTGCCTCGGTAGAGACTTAGCCAGCCGTGCCTGCCGGATTGCAGCCCATTAAGCAACAACCGCCCGGGGCCGATCGGCAAGTATACCCCTCGGCGAGTGGGAGCGGGAGTCCAAGTCAACCAGTGTAGCCTCGAGTGGGGCGGGTCCGTGTGCGTTCGCCCGGCTACATCAACTTGGAACTCGAAGTGGATCCAGTGTGGCGCAATGAACGTAGCGCGGAGCCGTCGCCGTGAGGCGCACAAGTAGACGGCCTTGAGCGTAGCGGGGAGCGAATCTAATTGACCTGTAATGTTAAGCTTCCAGAACCGTGATAGTCTCCGTGTAGCTGCTCGATGAGCGGGCAAAGTTGCGGGCGGGGGGCTTTACTAGGCAAGACCGATACCAGCCTCTAATGCGAGAGGGCCATAACCCAACAAGGCACCCACTAATCTGACTTCATGCGGGCGTACCGAGTTGGATCCGGCTTCCCCGAATCACACACTGTTGCATACCCGACGCATCAGTACGCCCCGGCGAGTTGGGCGATCCGGCTTCCCCACTACACTGTTGCAGAGGCAGCCTTAGTAACTGGCGGAGTCGTAGGAGACTATAGGTACAACCCCAGTGGAGGGGCCCTATTCGACCATAAGAGGCTTTGTGCAGCCGCGCCGGCGCTCGGTGAGACCAGTCGGCATTAACCCTTCTATAGGCCGCGGCGAATACGTCGCGCCGAATGCGTCCGTAAATAGTGTGTGTAGGACCCGGGTCAGGCATTGCTAGAGAGCCAGCAACAACCCGCGCTCCGGAGTGTTCTTACCTCCATACGCAAGCCGTTTGCATCCCTCATGAGTCCAGTCCTACTGTCTGCGCGCGGGGAGCACGGCGCATCCCGCGGTGCCGCCACGGCGAGGGCAAGAAGTACCTTCGGCTATGAACCTAGTGACGGGACGGGTGGGCCGGCGGCGCCCCCGGTAGLDLDV0300VOD150VIVOD913LL919099D100DLDOvi500001303lLlDLp0v?vOV1D0LDD050LD000V33o0VipvVoivVvvoVOLIVOv?vVDLDD300vV?oVD000lLDVLl50VD00L99D1000D00VIDvDvDVOD.LDVOIVVIOvov30I000090VVI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