GB/T 43166-2023 燕麥嗜酸菌西瓜亞種溯源檢測方法（正式版）

ICSCCS65.020.20燕麥嗜酸菌西瓜亞種溯源檢測方法國家標準化管理委員會IGB/T43166—2023本文件按照GB/T1.1—2020《標準化工作導則第1部分：標準化文件的結構和起草規(guī)則》的規(guī)定起草。請注意本文件的某些內容可能涉及專利。本文件的發(fā)布機構不承擔識別專利的責任。本文件由全國植物檢疫標準化技術委員會(SAC/TC271)提出并歸口。1GB/T43166—2023燕麥嗜酸菌西瓜亞種溯源檢測方法本文件描述了應用全基因組單核苷酸多態(tài)性(wgSNP)方法對燕麥嗜酸菌西瓜亞種進行溯源檢測本文件適用于燕麥嗜酸菌西瓜亞種的分子溯源檢測。2規(guī)范性引用文件本文件沒有規(guī)范性引用文件。3.13.33.4具有共同祖先的各物種間進化關系的樹。注：在進化樹中，每個分支節(jié)點代表其各分支的最近共同祖先，而節(jié)點間的線段長度對應演化距離。單核苷酸多態(tài)性singlenucleotidepolymorphism;SNP在基因組水平上由單個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)。該病菌的其他相關資料見附錄A。2GB/T43166—20235方法原理燕麥嗜酸菌西瓜亞種溯源檢測方法的原理是通過全基因組測序技術發(fā)掘物種的wgSNP位點，基于得到的單核苷酸多態(tài)性信息構建系統(tǒng)發(fā)育樹，進行聚類分析，從而對物種進行溯源歸類。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溯源檢測方法8.1檢測流程對燕麥嗜酸菌西瓜亞種進行溯源的流程包括菌株的活化、DNA的提取與純化、全基因組測序、數(shù)據(jù)8.2菌株活化在超凈工作臺環(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，對基因組DNA的提取方法見附錄C,提取后通過PCR擴增儀對其進行PCR擴增，并用電泳儀對PCR產(chǎn)物進行電泳，最后使用凝膠成像系統(tǒng)進行檢測。用核酸蛋白檢測儀測定DNA濃度及純度后，保存于—20℃冰箱備用。8.4全基因組測序以模式菌株(REF)或來源明確的菌株的全基因組序列為參考基因組，采用全基因組鳥槍法(WGS),利用測序儀對構建的PE(Paired-End)文庫進行2×150bp測序，測序有效深度(SequencingDepth)應達到200×,即測序所得到的堿基(bp)總量與待測物種基因組(Genome)大小的比值應達到200。8.5數(shù)據(jù)評估質控對待測物種測序的原始數(shù)據(jù)(rawreads)通過通用生物信息分析軟件進行質量評估。然后通過通3GB/T43166—20238.6序列比對按照通用生物信息分析軟件列出的流程開展序列比對：a)使用通用生物信息分析軟件將待測物種基因組有效數(shù)據(jù)與參考基因組(8.4)進行比對；b)使用通用生物信息分析軟件轉換比對結果的格式，并對其排序；c)統(tǒng)計比對結果；d)使用通用生物信息分析軟件進行重復序列的標注(MarkDuplicates);e)根據(jù)比對結果進行冗余序列(duplicatereads)分析、插入片段長度(insertsize)的分布等分析；f)利用通用生物信息分析軟件進行深度覆蓋率統(tǒng)計分析。8.7SNP檢測利用通用生物信息分析軟件的識別單倍型(HaplotypeCaller)算法分析待測物種基因組和參考基因組之間的基因型差異，對待測物種的SNP進行分析，得到變異信息并統(tǒng)計變異位點。將待測物種的全基因序列單核苷酸多態(tài)性位點按相同順序串聯(lián)起來，得到一條fasta序列(參考附錄D),然后將每個菌株等長的fasta序列輸入到建樹分析軟件，再通過系統(tǒng)發(fā)育樹鄰接法(NJ法)基于wgSNP構建系統(tǒng)發(fā)育樹，對菌株(含參考菌株)進行親緣關系分析。9結果判定根據(jù)待測物種序列在系統(tǒng)進化樹上與已知來源的燕麥嗜酸菌西瓜亞種的分支聚集結果進行判定：———如果待溯源物種與已知來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源地；———如果待溯源物種與已知來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源地；———如果待溯源物種未與已知來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源地。10樣品與原始數(shù)據(jù)保存分離得到的菌種轉移至LB液體培養(yǎng)基，在恒溫搖床中28℃培養(yǎng)36h,加入20%滅菌甘油，置于-80℃條件下保存至少12個月或采用無菌水保存法，嚴格防止擴散，保存期滿后，經(jīng)高溫高壓滅菌處理。10.2結果記錄保存和審核人員簽字。PCR產(chǎn)物凝膠電泳檢測需有電泳結果照片，測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)燕麥嗜酸菌西瓜亞種溯源檢測流程燕麥嗜酸菌西瓜亞種溯源檢測流程見圖B.1?；罨闐NA提取與純化全基因組測序數(shù)據(jù)評估質控序列比對結果判定樣品與原始數(shù)據(jù)及保存圖B.1燕麥嗜酸菌西瓜亞種溯源檢測流程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，置于恒溫搖床上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)將細菌懸浮液轉移至滅菌的離心管中，置于冷凍離心機中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，10000g離心5min,棄上清液。C.2DNA濃度及純度測定用核酸蛋白分析儀測定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)計算DNA純度，按照公式(C.2)計算DNA濃度：p=50×OD?60…………(C.2)p——DNA質量濃度，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，蛋白質的吸光度。PCR級DNA溶液的OD?60/OD?s?比值為1.7～1.9。(資料性)參考菌株wgSNP位點序列參考菌株wgSNP位點序列>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.LDVOIVVIOvov30I000090VVID01vOvpo000VLOD00090D0LLDD0ID0IV9OILI90IDI990LLLDVO9LI90990DDIVD00909V3p00VOIDVO99V9OIVVVII90VVOv9VI309VOIVOIVDOLI9013VOLLLIVOD0D09D00V9130V39VVI9D9IV9139013990VVIV9D0Lv300010001VODVO1lD150V301530130vovDDV3150DLVVo0vOVL13D1DLDVIvOV300005vv?vovVo5VOLOlLLL900009VlL9DVDovvovoivovVDb0VOl0500000909VvvovVDllIIV0ID1DVV.IVVVIIVDD09D?IIVOD00VDD0.MID0VDD0009D9009D.IVVOOILIDDVLDVIOIVOLO090099I39ID99909099ID9VVO909VIDVIS909V9DIVOID090D9VVOOIVOIVO99VVOVD99900D9919000DvvvVD19D0VVLIDDDLDDL919D009VpsVVOD02D099pVvDVI9DIvOVDIvVOllDoVVliVDiVD00Lp3V313L15000000000300VLDVIDLIVO5VDo3v33000vVovvs9V00VooV9DLD00000DLD00VLlLDD0V000300VLDD0000100L3900IVLDoVvoVDLp1V3o0LVvvovVVIDD0VD000VD.IVVOvOVVOD0IJIO?D0VIDLOIVOVID20000DVIDOLDI09D?IVVVDIIVD9390999V99909DVVI9D9D0090V?VVOVDO9II99ID90D03VOI3VDo0VOvVooVVIDVLI?VO990IVDOLIoI9DDLDIVL9D30DVODVI9D91D90VO9L39009L3D0ID90100DVLpDvoVDD0900D0900330900090D9099001b0v30033Dv3ovV330000V300390D0VD0D900VOl001500001300L00001VD010VD0LDVO0900V300ovVO0L900VDVLD0001ovVO50LVOVoDVVIDD00V9900ILDVLDDIVOVID0002.IIO0VVIDvDVVLD002.ILDO1DIIVD?L00VOp0VpoVo0IVVV9VDVLII99D9I9VVIDVOV99VVIVDD90VLIVLID99IDI09VOVVVD009V3999I90VOIVSVIo9VIDVLI9909IVV9DIvVVOD9VID99D009V3009VDIVD0DDLD0I9OLLIVDoVDvvovovVOllD030DLL1950L30VIv?vV33L1D0013L1300VD001D3DVDVlo1VoovvvVDD1DDDL9LDo1VLD019D01VDoIvVV30000LL150Vo0VLD000001301900VOIVDDVOllVOvVD0000IV150opV3D00VVDDOIIVOVovv?VOLD901DVO000IVVOIVOD?ILD0090LD?1D0000ID0000000DVOVOIDVL999V9900VOv?VVOD90IIO999000090990VDD0DDIDDIDVID0900990909IO90390I9DLIpvvVDDDL99L390VDI399VVDIIv9VD0D0IVI999VVOVooVp9vDvDVOI99DL009150lLlvVV033033DVDlivVD0lllb0V3DllV3svovpvvV390LLllDLD09D1oVVD010V000VIvOVDDLVLOD090000D9000VIVVDVi?VOIVL30001001VO0000DVOvDVOo0VDD19LL999VVILIIIO9000999VVVVDOIDI99VLLIDVD9VOVOVDI909VOVoVVLI300VODVIIO9DLvD920L999900V593LIVIVDDVOLI0939IVIvVVD9I3LDIVIVOI3IDLIp0VDDLIIpVVL909VLIV300150V3300900vv3ooVviD90VL?V3o9V?vIVLlIVD001990VDVIvvovov9v3voD010DDVDIVL?V9000VoovDVOL1o9vovovVO0D0090ovVo0VOIVO09Vo3VIVLD001D00VD00090DVIV30DI9DD0IVIOVOVVVO9VDIVOIOVVI3pDVVDVII99D9VD9I90VOVOI39I9I39DDL9IVOD01D0150VDLIVD1301vvVVIDL30VODLIVIVL1p0vvVDo1V300991DvDV300000V100IVDoVODl?DVDo00000901000090000IVD00000000VDVL?VD0VD309300VVDVO00LI50DI90V909DVOLI9VIVOLIVO99900VO99990V399VVOLIVO99VVOVVV39DVI3DDIOVD09LD00000Vop300D19llD1500DLlV3ovovvVDDDVOvpp0019DD9013039090D10vpDVDoDV10DV913VoDvpvvovpoVo9VOLllLD0090Lp0vovovVLOl0VovDovDo1vO0150DL1p0vo05000900vVvD00LLD09VVID1D0lIVDovoVD0150000VIVo0vOV9001V0o0VOL1VLD090V0IDVpDVDLID0009V0D09vOv39?VVLDD.LIVV?pVVODVDooVOID0DVOvopp00DLIID.L200SIOVVDDIV9DIDDI9DVODIV9OVVD0999ID90000I9ID09009990LIVLID09VVI9V9p90DVGB/T43166—2023GACTTCGTAGCTGAGGCTTGGGGAACGGTCCGCTTAAGCAGTTCGCACATTTCATAGATATACGGCCCGATATTCAGGTCGCGGTGTTGGACCTGGCAGCTTGGGTCGTGTACAGGTTCACGCAGGCCCTGCCCGAAGGCGGGGAGGGCCAGTTGCTGATCGGCTCTGCAGGGGGGATCACAACCATCGTGGAACACAACGGCGAGCGAGAACGCCAACGGGAAGACGATAACCAGGCGAACCAACCGACCAGAATGACTTATAGACAGTAGGGTAAGTCGCGCGAGAGTGGTTACCGACGCCG