GB/T 43166-2023 燕麦嗜酸菌西瓜亚种溯源检测方法（正式版）

ICSCCS65.020.20燕麦嗜酸菌西瓜亚种溯源检测方法国家标准化管理委员会IGB/T43166—2023本文件按照GB/T1.1—2020《标准化工作导则第1部分：标准化文件的结构和起草规则》的规定起草。请注意本文件的某些内容可能涉及专利。本文件的发布机构不承担识别专利的责任。本文件由全国植物检疫标准化技术委员会(SAC/TC271)提出并归口。1GB/T43166—2023燕麦嗜酸菌西瓜亚种溯源检测方法本文件描述了应用全基因组单核苷酸多态性(wgSNP)方法对燕麦嗜酸菌西瓜亚种进行溯源检测本文件适用于燕麦嗜酸菌西瓜亚种的分子溯源检测。2规范性引用文件本文件没有规范性引用文件。3.13.33.4具有共同祖先的各物种间进化关系的树。注：在进化树中，每个分支节点代表其各分支的最近共同祖先，而节点间的线段长度对应演化距离。单核苷酸多态性singlenucleotidepolymorphism;SNP在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。在全基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。4病菌的基本信息Pseudomonaspseudoalcaligenessubsp.citrulliSchaadetal.,1978单胞菌科(Comamonadaceae),噬酸菌属(Acidovorax),西瓜亚种(Acidovoraxavenaesubsp.citrulli)。该病菌的其他相关资料见附录A。2GB/T43166—20235方法原理燕麦嗜酸菌西瓜亚种溯源检测方法的原理是通过全基因组测序技术发掘物种的wgSNP位点，基于得到的单核苷酸多态性信息构建系统发育树，进行聚类分析，从而对物种进行溯源归类。6仪器和器具种针。7试剂和培养基溶解后，每份10mL分装到玻璃试管中。以上培养基均在高压灭菌锅中121℃高压灭菌20min,备用。8溯源检测方法8.1检测流程对燕麦嗜酸菌西瓜亚种进行溯源的流程包括菌株的活化、DNA的提取与纯化、全基因组测序、数据8.2菌株活化在超净工作台环境中，将保存的菌株用LB固体培养基用接种针取菌采用平板培养皿上划线法活化，于28℃恒温培养箱中倒置培养2d～3d备用。8.3DNA提取与纯化使用CTAB法或商业DNA提取试剂盒，对基因组DNA的提取方法见附录C,提取后通过PCR扩增仪对其进行PCR扩增，并用电泳仪对PCR产物进行电泳，最后使用凝胶成像系统进行检测。用核酸蛋白检测仪测定DNA浓度及纯度后，保存于—20℃冰箱备用。8.4全基因组测序以模式菌株(REF)或来源明确的菌株的全基因组序列为参考基因组，采用全基因组鸟枪法(WGS),利用测序仪对构建的PE(Paired-End)文库进行2×150bp测序，测序有效深度(SequencingDepth)应达到200×,即测序所得到的碱基(bp)总量与待测物种基因组(Genome)大小的比值应达到200。8.5数据评估质控对待测物种测序的原始数据(rawreads)通过通用生物信息分析软件进行质量评估。然后通过通3GB/T43166—20238.6序列比对按照通用生物信息分析软件列出的流程开展序列比对：a)使用通用生物信息分析软件将待测物种基因组有效数据与参考基因组(8.4)进行比对；b)使用通用生物信息分析软件转换比对结果的格式，并对其排序；c)统计比对结果；d)使用通用生物信息分析软件进行重复序列的标注(MarkDuplicates);e)根据比对结果进行冗余序列(duplicatereads)分析、插入片段长度(insertsize)的分布等分析；f)利用通用生物信息分析软件进行深度覆盖率统计分析。8.7SNP检测利用通用生物信息分析软件的识别单倍型(HaplotypeCaller)算法分析待测物种基因组和参考基因组之间的基因型差异，对待测物种的SNP进行分析，得到变异信息并统计变异位点。将待测物种的全基因序列单核苷酸多态性位点按相同顺序串联起来，得到一条fasta序列(参考附录D),然后将每个菌株等长的fasta序列输入到建树分析软件，再通过系统发育树邻接法(NJ法)基于wgSNP构建系统发育树，对菌株(含参考菌株)进行亲缘关系分析。9结果判定根据待测物种序列在系统进化树上与已知来源的燕麦嗜酸菌西瓜亚种的分支聚集结果进行判定：———如果待溯源物种与已知来源物种序列聚为一支，支持率为100%,即可判定待溯源物种与已知溯源物种具有相同的来源地；———如果待溯源物种与已知来源物种序列聚为一支，支持率低于100%,即可判定该物种与已知溯源物种疑似为相同的来源地；———如果待溯源物种未与已知来源物种序列聚为一支，则判定该物种与已知溯源物种不是同一来源地。10样品与原始数据保存分离得到的菌种转移至LB液体培养基，在恒温摇床中28℃培养36h,加入20%灭菌甘油，置于-80℃条件下保存至少12个月或采用无菌水保存法，严格防止扩散，保存期满后，经高温高压灭菌处理。10.2结果记录保存和审核人员签字。PCR产物凝胶电泳检测需有电泳结果照片，测序全基因组序列需要保存电子文件。4GB/T43166—2023(资料性)A.1分布var.saccharinus)、黄瓜(C.sativus)、南瓜(C.moschata)、西葫芦(C.pepo)、番茄(LycopersiconEsculen-A.3为害症状5GB/T43166—2023(资料性)燕麦嗜酸菌西瓜亚种溯源检测流程燕麦嗜酸菌西瓜亚种溯源检测流程见图B.1。活化菌株DNA提取与纯化全基因组测序数据评估质控序列比对结果判定样品与原始数据及保存图B.1燕麦嗜酸菌西瓜亚种溯源检测流程6(资料性)基因组DNA制备C.1DNA制备使用下列CTAB方法提取病菌DNA,或使用商业化试剂盒提取DNA。a)菌株的培养及DNA制备的前处理。1)将菌株接种于装有LB液体培养基试管中，置于恒温摇床上28℃摇培28h。2)在试管中加入0.1mol/L磷酸钠缓冲液10mL洗下菌体。3)将细菌悬浮液转移至灭菌的离心管中，置于冷冻离心机中12000g下离心15min,弃上清液。b)DNA的提取。37℃置水浴锅中水浴1h。2)加入等体积的三氯甲烷/异戊醇(24:1),混匀，10000g离心5min,将上清液转移至新的灭菌离心管中。3)加入等体积的酚/三氯甲烷/异戊醇(25:24:1)混匀，10000g离心5min,将上清液转移至新的灭菌离心管中。4)加入0.6倍体积的异丙醇，轻轻混合至DNA沉淀下来，10000g离心5min,弃上清液。C.2DNA浓度及纯度测定用核酸蛋白分析仪测定DNA的纯度与浓度，分别取得260nm和280nm处的吸收值，按照公式(C.1)计算DNA纯度，按照公式(C.2)计算DNA浓度：p=50×OD₂60…………(C.2)p——DNA质量浓度，单位为微克每毫升(μg/mL);OD₉260nm处吸光光度值，核酸的吸光度；OD₂80——280nm处吸光光度值，蛋白质的吸光度。PCR级DNA溶液的OD₂60/OD₂s₀比值为1.7～1.9。(资料性)参考菌株wgSNP位点序列参考菌株wgSNP位点序列>REFCTTTTAGTGATTCAACTGGGACCGTCAGGCCTCGCCGATCGTTCCGGTGGTGAAACTTCATGCGTGGGTATCCGGCTCTATCGTACTCCACTCGACTTAGTCGGGTCGGGCCAGGCCTCACGTTCCTTAGCGATGTTGAATCTCCACGTGCTGGGTGTTTCGCGGGCCCGCGGCATGGTCTGTGCTCTAACGGCGTCACGACAGCCTTGTCTGACTGGGATCAGGGGTGCCGGTCTCGGACCTACTCACGTGGCCGTCGGCCACAITCGACGCACGGTGGGTATCCGTCCCAGTAGCCTATGCACGCACTTTGTGCCGTAGGGCGGGGCGGTGTTGCGGGCGGAGGGCGCIGGACGGGTGGCACGCGGGTGCATACTTCGCIATGGTAAGCTGATGTTACACCGTIGCCTGTAGGCAAGGGTGCCGGAGTCTGCCGGACGAGCGCCCGGGGTTTCTGTTATAATCAGCGGCGATCGCCCTGCCGTCGCACTGGAGTGCTAGTAGGGTCTGCCTACATAGGGAATTTTGAACGCGGATTCGGACGTGACCAGCTGGTCTCCCATACCGCTCGTAAAACGGCGGCTGAGCCCGTGGCCGTCCTCCGCGCGATCCGGCCCGTCGCCTCGTCGTATCCGGCCTTATGGGGAGGGATCCCCGCCTCTCCAAGGCGCCAAGTGGTCACCACTGTCCCTGCTCGGTCCCCCCTCATCGGCCGGGCCIGCITGCGCCCTItTCCGCAI'"TGGGATAGCGGGTGAGCITTCCTTCCGTGCGGCGCTTGCACAGGAAGGGGTGGAGTCTTGTTATCGGACTAGTGAATAGTGCCCGATCCTGGATCGATTGCACCAGCTTATGAGCTTGGGATCGTGAGAGGCGAGGACGGTCCACCCTCAGGGCCGCAAGTTTGTATATGTCTGGGAGCTCACGCAGCGCTACGAACGGCGGTAAGTAAGGAGGGGCACCCTGACCTGAGTGTTCGTGCCGGATGCCCACTACCCTTTCCACCAGCTTCCCCCCAGATGTGGCAGAGACTCTGTTGCTTTACTGTGTTrTCCTAAGGAAGTCATCGGGAATAACGAGATGAGACCATGTCTCGTCCACGATCGGCCGCACGCTCCCCAGTGCGACGGGTGCCGCGTGGGAGCTGCGAGAACAAGGCGTTCTGGTAAAGACCGACTAGCTGGCCGGCGTTCTGGCGCGCCGGCTCCGACACAGCCGGTCGCACGCTGATCGACAACGTTGCCTATTTTCCAGCCTGCCTGACACCAGGCAGGATTGCACTTTGGGTGCGGCGCATCTGGCCCCCTCCCCTGATCTCGATGATCATACCAGCTTTGCACATCGCACGCGTAAAGGCTACCGCTGTGGCCAGCCCCAGATCGAAGCGCCGGTGTACGCTATCAAGGGCGTTTICACATGTCTGCCAGAGGTGCTCGGCTGGCGGCCCGACTACATAGCCAACGTCGACTCAGACATTGGCAGTGCCTCTCGGCGCTGCTTGTCGTTCAATGGCTCGCCCGCTATTGGGACAACCTGCGGTCAGTTGTTCTAGCGGCTGCAAGTCGGACACCAGTGTGCGGTCGCGCCTCGTCGCCTGACACCGATGTCACGCGTTTTCCCGGAGGCAACGTGTGGTGTGTGCGTAATGCTCCTCGGGAATGGAAGTGGGGGCTGCGGACCGCCCCGCACTAGACGTGACCGGCTCTGTGTGCGACCTTGTAGTGAGGTGCACCCAGTGAGTTGGCTGGGTCTCTGTGTGGGAGCTGCTTTAAACGTGTAGTGCGTACACGCCAGATCCATCAAACCCCCAGATGCTGTGCCGCTCGGCGTCTATGGTGAGCCAGGGACGCCGGGCGTGGGCCGCTCCGGTGCGAGTCTGGCTCCGCGCTTCTCGGTATGCGCCTCCTGAACGGCTGGGCCCGACGATCGAACACCGACGCGGAGTGTTGTGTGCACCCTAGCCCGGCCGTAGTGCCTCGAGCCTTGAAGGCGCCTTGGTTAAGGCACAGCTCTGAGCCCTGTCCTGTTTCTGTGTGCGTCCGGCCACGATCCGAAACACCGGGGCGCGTGATCCGCCGGGGTCCTTCGCGCCGCGCGTCCTATCAGCATCACTTGGACGCGGAACGTGCATCGCTCACACTGATTCACAGGTTGCCGCACTGGAGCCCCCGACGACCGCCGGGCAAGAGGTCAGCATAATCGCCCAGGACGGCCCGTCTGCTATCGGCGGCTGTGGTGTTGTTCTTTTTCCGB/T43166—2023TTGCCGCCCTCGGAGCACTCCCGAGGCATGGCAAGATGTAGTACTCTCGAAATAGCAGCTCTACTACGATTACGACGGTTTCTCCCcCCGTGGAGCGGAGGCAAGCGAGGTTCTGGACGGCTGCTCTTCGGAGGCGCCCCGGGTTACTCCGTCGTGAGGCGTAGACAGCCACTATGGCTTATCGACAACTTCCTTAATTTTCCTCCATAGCCGTCCGAAGTTCTAATACAACTGCTATAAATCCCAACCTACCGGGTACACATGTTGGTCGTGTTGTAACTTAGGCGATAGTTCGTTTGATGTAGTATTCACACTTTTCGTCAACCCCGACGGGCCCGCGTGGGCGACCGGGGGCGCCTCGGCGCCGCCACAGAAGCGTCCGGGGGGTCCCGGGCTTGCTTAAGCCATACGGGGTGTAGTTCTCCCTACGAAGCTACAGGCTTTTCCAGCCACTGTTGCATAGTTACCAGATTAAGCCTTCACAAACGCCGTGCTCGCGACGCGGGCCACTGCGTCGGACTATCACTACCTTGTTATATCCGAGACGCAACGAAGCCCGCACGCCGTTAGGCAATGGTTTTCCGCAACGCCCCGTAAGGCCGTACCATAGTCCGCGCCTTCCGCCGCCGCATCAGAGAGGGCGATCCGCCTGCGAGGTGAGATTGGGGGGCAGGGCGATGCCCCGGGTGCGTACTTGGCACCGCAGATACTTCACATTTGTTGTGCGGCAGCCCTTAGTCACCAGGTTGTCGACGCGGTCATGAAGCCGTGCGCCACCTGGGCCCACCCGCACGCCGGTCACATGCGCGAAGGTGGTTGCCGAGCGCCCTTGGCGGGCAACATACTTCCGCCCTCCCGGCTCGTGCGTGTCGCCCCTCAAGACGCACTCACAAGAGAGTCCGCCCCTAATCTTCCCTGTCAITATACCCCTATGGTCACCCCTAGCAGCGTATTGAACAGTGGTCACCGAGGAGGCCGCGTGTCCGGCGCGGACCCCGCTATACTTTCCACCTCCTTTGGTTGGGATGGATGGCCTTTATACATTCGCGGTTACAACATCGTTAGGTGCGTCGTCACCGTATCCTGGCGAGCTCACCGGCTTGGCCACTCTCGGTCGGCTACTCAAATTAATCACCGCCCGACCACGACTCTCTGGGCCAACCCGGCTGTGGGCTTTGGTCATAAACATCGAGGTGCACACTAAGGCTATAGGACCGCAATTGAGGCAATCAGTCTGACTTACCGCTCTCGACCGTAGCCATCAITTACCCTGGGTACTTITGTCATGTGAACCCCCCGTCCTCCACCCGGAGGATTTGGCGGAACTGTCACACCCTATTCACACTCGGCTCCGCCGCGGGCAATCCCAGTGAGAGCGCGGCCTGCATCCCCTTGGCAATTCAAATCTGTGAGCTCATCCCATCACAGCCGACGGATGGTGCCCTTTTGCTGTTTGCACGGCCTGTCACCTGGCGTCATTTCCATCACATCTAGTACTCCGCCAGGGCGGATAAGAGCGAACTCCTTAAGAACTGCCCCCGGGGCTCATCCCCAGGTGGCAGGTCCTATAGTGGCTCAGCGGGATATAGCAGGAGCCACTGCTAGATAAAGITGTTGAGGCTCTGACGCTTACCGCCGGCGCGGTGTGGTGTCGACGCGGCGCTTGTGCTCCCCGGTGCCCTCTCGTCGCGTCCCCGCCGTTCGGCAATGGGAGGCACGGGAGCGCTGGTCCGCCCAGTTCGGGAGTTGCGTTTGCCGGGGGATGATACGGCCACCACGGACCCATTTGGCTAATATGAGCGGCCAAAGTGTCTTCGTCCTATGCCTGCCTGGTGGTGCGATGTACCGACGCTTCCTGCACTTGACTGAGATATGCAGcCGTTAGCCCGCGTGGCCCACGTCCITrICACTGTAICTGCCGAATCGCGTCATCTACGACAGTAATTGICGACGAAGTGGGACAGTACATATAGCCCTTTTACAGGAGAGTCCAATGACTACACTCTTCCCAGGACCGGAGATGGCTTCTTGTGGGGCCTCCTACCCCTGGCCCCCTCACACCGCGCCGAAGTTTGGTTAGATTGATCTAAGGCCTCGCCCCACGCCGCGCTGGTCGAGTTGAGGGACCCATCCCGTCGGTCGGCAACATGAGACATTCGACGTTCTGCCTTCATGGCTACAAAGGCGGTTCTTCAGTCAATCCAACCTTCGCCCAGGACCCAGGTGCAATAAGTAAGCTGGCGGTGAAGCCGCCCACGTGCCCGGATCCGCCGCCCGGCGGTCCCAGCACGTAATTGAGGAGACGTGTCCCGCGAGCGTGATCGGTCTGGCCTACCTCGGAGCCCTCCTTCGTATCACAATGCAAGTAACTGCTCGACTTGTGCCCGAGCTCAGGTATTCGCGCACCCCCACGGGAGTACAAGGCGAAGGCTGCCCTTACCTCGAGAGCGCTGATTTGCACCGTGCGGTGGCGACAGTCTTTGTTAGTAGGGGCGCCAACATCGGCACCTACTGGTCATGTTTCCACCGCGCGAGAACACAGTGCGGGTTATCACTGGTTCCGCGCTAACGCCGCTAACAITCICCGCAATGACCCGTTGGCTTIAAAACGCGCGAACAAGGTGGCACGTGTAACAGGTAATACGTGATTGCCGATCTTCCGCGCGTTGCCATGGCGAGAGCGGGCCCATTGGCATGATTTCCCCGGTCCGCCTGGGAGGGGCGGCTGGGCGCCGGGCGTTCCCGCTCCCAGGCTACACCGGACGCCAAACGACATTCTGCGCGCCGCCGTCCACCCCATTGCTTACTCCGTCCGAGCGCCAGGGCCGCGAGGCATGCGGAGCTCAGGTAGATATCAACTCTCAGGGGCTCCCCGCGGGOIVO9IIVD00IOVDVVVD999VDvVO99VIVOp3Vo0D9090I9VVIIDIIDVII009VLɔDVODI9VVDloVD100300vov000DL3oVviv391ovvvɔp1V3Lo199ovVDDIVlIVO139D00VVlLD91VDLVO000VvoVODLVLIVDo0VOOVDo0VDLLDL9D00vov30000LVD0V000V90VVi3000L00V!009000VO.IVDLVODL₁D30vVo9V39VO00VvɔV0p0VVODIVOLLDVOD30V9000D00LOV300VOVID9000IVODVIOVVOD00VOVO99IVDV90090099ID909090D0LID00D00IV90D909VD0DV9DIOIIVDOVDo0VODIIDIOVD99LI90009V9D9009VD9VDDDI9VDIVD009LDIVOOI0IDLDO1vOVDIVDD130VlIVDLDVOl3VD9DVD3913091VD3VDVID1VLD101313090V9130131D30D001001V300L⁵IVVLLlL3000000VDivVOVi5L000009VivVOD0090VO⁰!)L1090V9!)L1001D0VDpDVDo.IVɔvvovDvvVpoVD0D09VVDLDIDLLDIVD00LDDVVVIDVOvvVDvovVoovVVD9V39VD0V9990VIDV9VDoVV99IVODLIDI09DIDLID9VD0DVIO90VOID00V999090VOVVIII9D999V9009I39V9999VD9ID9IOVDI9OVIVD3VOI99ID99VVD9VID39D9V9909VDDpDVLɔvVVD9DVVD139VVOD39D1910D00300D9D0990vvop0VooVopDVDVDDLD90Vip0V5lDV1501vVVooVVIDIVoovvvVO0010VODVV3oovvOV1povvo1vovovvVIVVooV3l00Ll)13VDD₁00VLLLDVLIIVDvvvVvovvpVLɔVVD0D0vVVLOVIVVIVOVDLDD1V300IVVLVOL00VVO09VIVVIDLIS0IDOV9909VOVVIVOvVp0VVIIDI909999VID0VVVI90DIDDI3D09090VviDDLLl19DVVLLL33L3lIVDD09lLDL3DIVODL3313DlLlDViolIVOl1009150ll91903020VIVOD00llDOVLpV301300v3o5V15ovVO00050LLl5V50000010LDo0VO0IVDDLlDVpoV!ILD00vvV30p01D00vɔV0900005DvDVD00030pVDD9000DL₁D90DLID00200001DVDoVpVOIVIVODDLDD2IDVOVD50VOOVOVID₉IDID02DOVDIDOVVOOIODVOD0VVIDI3D00IOVO9DI900ID0II9DV999999LLDDVvOVo0V3090VDL9009LLISLIOVLI5DVVI90VvOVOVLD9VOL9D1909VDD991oVVIVIDDL1D0010190130VvD9230V0o9vɔV33900v30009vOVDL3000VIDVVDDLIDLID09V9D009LO99LIDOILIVIVLDDIDDVID9992IDVLIDOLIDVO9IDLIIDDVVDSV9VVɔvVVID9I099IVVOIVVIVI900VLD99ID0D09DIVOVIVO90VID99D99V999VO09VDD0219D00D1vDD0DD9DllLll9D1DD9D9V3sVDVOD00D90DD0D1VVLD0D00v3OVLlovpovvDLLDD00L09DLIDLD90000001D9L9DVVpoVD01D0DLDV39IVIOVD001D01501D90VD001D9V3o0VO990I9OI09I9VO90090990D932LD9VD9090099093990VO9D₉IDI90VIDVOID01DVL5000I300900LD0LLDLD000001500000VODLDOVIVOVOD0VɔvVIVO0D₀ID909V000VI9999VIOIO0V9D90I99009VO99LD99IOVOLIVO9I9099IVOOIDI0VVVOVDID00VID900I9OVIVD9V3OIVIVIVOVVD9IVOIDV9DI90ID9VO99IOIIVVIDVIVD90ID9VVD9I9VDOLIVD0VDD00D0019VovvIVDl130IvVDVDDOIV139DVIDLLll9VVllVVlLD13VLɔvVV13009VOLIVL10VoovDVDVIVODLDDL5001LpVVlIVLLIV3o1VO0900vovooV5090V5000LD00000001ovVOLDD0D0IVD000000voVD30LD000000330V.LLDDLDD00001D0VVOD.L₁D₀1000VDoVD0VODVLID90VoDVVDOID9IDOVOOI3099VOvDo0VOIVOVI50VD99990IVOVD999VIVV99DL3DV9509I99900VɔVVIVVO99VVODDIVO90IDIVOIDIVIVVDSVDODV990990909I9D0VD9090V3D00D9VDoVVDL3D01llL39VD019DDD31D9130DD13309DVL3D0D9990030D3L3VLDVlL000D0000VIvOVIVLD000D0EVID0VID01vVVo010Voovɔv0000001901o3VOLDLD0GB/T43166—2023GTCGGCAGCGCGGGCGGTACTGGGTAGCTGCAACGCTCGGTCATACCGGTAGATGAGCACCCAAAGCATCATAAGAACGTAGACAAATTCTGTTTCTCCTCAGAATGGATGCTTAACCTATAACCGTACCTTTTTAGATCCTGGAGCCTTTTAGTCGGGGGGGGCGTTACAATGGGGCCAGGTTGGGAGTAGGGCGGCAGAGCCGCGGGGCGCGGAGAGTCGACGGCGCTGTCCATTGTCATTAAATACTAACTAGTTCCGGCCAGTCGGGACCAAGGGGACACCGCGGACAGAGGCAGGACTGAACGGATCGCCGAGCAGTAGCATTAGCACCCTTGGGCTCGGTCGAAGAGTAATGTTCTAAACACATCCCTCCGTCCAAGGTATTTGCGTCGGCGGCCCACGCGAGGTCTATTTGCGGCTATCTGTTCCGCACCCAGCCTCCTAAGTAGTCCTAGTGACACCTCCCGGACCCGAATCAACTCGGTAGGCAAGTGAGTTGGCTGGCGTAGATACCCTGGATTATAGGCCGTGGAGGGATGCGATGTCGTTTATGCCCCCCAAGGATCTCGGCACGTGGGATCGGAAGAGGTCGCATCCCTCGCTGGACCCGCTTAAGCGGACTTAAGGGGTGGGTCCACTGCCGTCCTCGCGTCATGTGCCGGCTGGTCCGTGTGACTCACCGCCACCCCCGTCGACCAGCTGATGGGTCGCGTTATTGTGGCCGCAGCCCACTATCATGGGCGATGACACCACAGGCGGCGTAAGTGCGTGTTGCAAAGCGCGTCATTGGACCTTTTCTCCTACACCTTAGTGTCGTCGAATACGGGGCTTCTCACCGTGTAGGCCTCCGACGGGAACCTTCGTCGCCTCCGGGTTTGCGACTCATGGTCGCGTCCGGAAGCGCCTCATAACTTGTACCTATACAACACTACACCGCCTCCGCCTATCCTCTTGTTTAGAAAGCCAAGTCATCGTACCGCGCCTCACCGCATCCATGGCGGATAGCCTGTCAAGATTGGCTACGCGGGAACAAGGTTGTGTCCCATCCTAGGTTGTAGCTCGGACGGCTCTTGAGATCGCCGTTGCACTGGACGCGGGCCGTCGTCGCCGCTAGCGTCCAGTGATCCAGCCGCCGGGCGAAGCAGGTAGGTCTCGGCCACACGCACATCCTCGCCGAGCGTACGGCCCCGAGAGGCCAGTAGCGTGCCGGTCTCATGGAACAACGTGCCTCCGCACGGCGTCACCGGTCTGCTAGTCAGTACGAATCCCGTCACGATTTGACTCCTCGGGCCGTACGTACCGCCAGGACGTGGGATTCACGGGGCGAGGGAGTAGGGGACGCACGAAGAGTCAGCCTCTGGCGTTGGGGGCGGTAGAGGTAGACGACCATCAGTGGCTGGCCATCGCATGATTGCCGGTCGCGCACCACGTCCGGCAGCCGGGTACCCCGCGACCCGGGACGGTCAGACTCGGGCTCCGAGAGAACACGTTTACGCGGCAGCCCTCCGTCGAGTTACTCACCCCCGCGGGTGCGTCGACGGGGCGGAGCGAGCGTGACGTGCCGGTGCGTCCGCGTCCATCTGGTCTTCACAGGGGTTGGAGATTGGTATCGTACCCACGAACTCGAACAAGGCGTAAATATTTCTAGAAAACTCCTAGCAGAGTTCGAAAAAATACGCCCCTAGCGCGATCCCGCCCAAAAGCGGTGAAACACGCTCCTGCTAGTAGCCCACTTCTTAATCGTATTGAATCTACACTGACAGTGACCACGAGAAAGTTCACTGACCGAGCTCGAGTCGCCAGCGCCTTCTTGGACTAGGGGCGATTGTAAAGTTCCCTAAAAACCCAAAACGCCGCGCGTGGTGTCGACCGGGAATCGTCGGGATACGTGCGTTTTGTGGCACATGACGGATCCCTTAGGCCCATGACCTTGCGAAGCTGTTCACCACGAAGTATGGGCGCTGCCTCGGTAGAGACTTAGCCAGCCGTGCCTGCCGGATTGCAGCCCATTAAGCAACAACCGCCCGGGGCCGATCGGCAAGTATACCCCTCGGCGAGTGGGAGCGGGAGTCCAAGTCAACCAGTGTAGCCTCGAGTGGGGCGGGTCCGTGTGCGTTCGCCCGGCTACATCAACTTGGAACTCGAAGTGGATCCAGTGTGGCGCAATGAACGTAGCGCGGAGCCGTCGCCGTGAGGCGCACAAGTAGACGGCCTTGAGCGTAGCGGGGAGCGAATCTAATTGACCTGTAATGTTAAGCTTCCAGAACCGTGATAGTCTCCGTGTAGCTGCTCGATGAGCGGGCAAAGTTGCGGGCGGGGGGCTTTACTAGGCAAGACCGATACCAGCCTCTAATGCGAGAGGGCCATAACCCAACAAGGCACCCACTAATCTGACTTCATGCGGGCGTACCGAGTTGGATCCGGCTTCCCCGAATCACACACTGTTGCATACCCGACGCATCAGTACGCCCCGGCGAGTTGGGCGATCCGGCTTCCCCACTACACTGTTGCAGAGGCAGCCTTAGTAACTGGCGGAGTCGTAGGAGACTATAGGTACAACCCCAGTGGAGGGGCCCTATTCGACCATAAGAGGCTTTGTGCAGCCGCGCCGGCGCTCGGTGAGACCAGTCGGCATTAACCCTTCTATAGGCCGCGGCGAATACGTCGCGCCGAATGCGTCCGTAAATAGTGTGTGTAGGACCCGGGTCAGGCATTGCTAGAGAGCCAGCAACAACCCGCGCTCCGGAGTGTTCTTACCTCCATACGCAAGCCGTTTGCATCCCTCATGAGTCCAGTCCTACTGTCTGCGCGCGGGGAGCACGGCGCATCCCGCGGTGCCGCCACGGCGAGGGCAAGAAGTACCTTCGGCTATGAACCTAGTGACGGGACGGGTGGGCCGGCGGCGCCCCCGGTAGLDLDV0300VOD150VIVOD913LL919099D100DLDOvi500001303lLlDLp0vɔvOV1D0LDD050LD000V33o0VipvVoivVvvoVOLIVOvɔvVDLDD300vVɔoVD000lLDVLl50VD00L99D1000D00VIDvDvDVOD.LDVOIVVIOvov30I000090VVID01vOvpo000VLOD00090D0LLDD0ID0IV9OILI90IDI990LLLDVO9LI90990DDIVD00909V3p00VOIDVO99V9OIVVVII90VVOv9VI309VOIVOIVDOLI9013VOLLLIVOD0D09D00V9130V39VVI9D9IV9139013990VVIV9D0Lv300010001VODVO1lD150V301530130vovDDV3150DLVVo0vOVL13D1DLDVIvOV300005vvɔvovVo5VOLOlLLL900009VlL9DVDovvovoivovVDb0VOl0500000909VvvovVDllIIV0ID1DVV.IVVVIIVDD09D₀IIVOD00VDD0.MID0VDD0009D9009D.IVVOOILIDDVLDVIOIVOLO090099I39ID99909099ID9VVO909VIDVIS909V9DIVOID090D9VVOOIVOIVO99VVOVD99900D9919000DvvvVD19D0VVLIDDDLDDL919D009VpsVVOD02D099pVvDVI9DIvOVDIvVOllDoVVliVDiVD00Lp3V313L15000000000300VLDVIDLIVO5VDo3v33000vVovvs9V00VooV9DLD00000DLD00VLlLDD0V000300VLDD0000100L3900IVLDoVvoVDLp1V3o0LVvvovVVIDD0VD000VD.IVVOvOVVOD0IJIO₁D0VIDLOIVOVID20000DVIDOLDI09D₀IVVVDIIVD9390999V99909DVVI9D9D0090VɔVVOVDO9II99ID90D03VOI3VDo0VOvVooVVIDVLIɔVO990IVDOLIoI9DDLDIVL9D30DVODVI9D91D90VO9L39009L3D0ID90100DVLpDvoVDD0900D0900330900090D9099001b0v30033Dv3ovV330000V300390D0VD0D900VOl001500001300L00001VD010VD0LDVO0900V300ovVO0L900VDVLD0001ovVO50LVOVoDVVIDD00V9900ILDVLDDIVOVID0002.IIO0VVIDvDVVLD002.ILDO1DIIVD₉L00VOp0VpoVo0IVVV9VDVLII99D9I9VVIDVOV99VVIVDD90VLIVLID99IDI09VOVVVD009V3999I90VOIVSVIo9VIDVLI9909IVV9DIvVVOD9VID99D009V3009VDIVD0DDLD0I9OLLIVDoVDvvovovVOllD030DLL1950L30VIvɔvV33L1D0013L1300VD001D3DVDVlo1VoovvvVDD1DDDL9LDo1VLD019D01VDoIvVV30000LL150Vo0VLD000001301900VOIVDDVOllVOvVD0000IV150opV3D00VVDDOIIVOVovvɔVOLD901DVO000IVVOIVOD₀ILD0090LD₀1D0000ID0000000DVOVOIDVL999V9900VOvɔVVOD90IIO999000090990VDD0DDIDDIDVID0900990909IO90390I9DLIpvvVDDDL99L390VDI399VVDIIv9VD0D0IVI999VVOVooVp9vDvDVOI99DL009150lLlvVV033033DVDlivVD0lllb0V3DllV3svovpvvV390LLllDLD09D1oVVD010V000VIvOVDDLVLOD090000D9000VIVVDViɔVOIVL30001001VO0000DVOvDVOo0VDD19LL999VVILIIIO9000999VVVVDOIDI99VLLIDVD9VOVOVDI909VOVoVVLI300VODVIIO9DLvD920L999900V593LIVIVDDVOLI0939IVIvVVD9I3LDIVIVOI3IDLIp0VDDLIIpVVL909VLIV300150V3300900vv3ooVviD90VLɔV3o9VɔvIVLlIVD001990VDVIvvovov9v3voD010DDVDIVLɔV9000VoovDVOL1o9vovovVO0D0090ovVo0VOIVO09Vo3VIVLD001D00VD00090DVIV30DI9DD0IVIOVOVVVO9VDIVOIOVVI3pDVVDVII99D9VD9I90VOVOI39I9I39DDL9IVOD01D0150VDLIVD1301vvVVIDL30VODLIVIVL1p0vvVDo1V300991DvDV300000V100IVDoVODl₁DVDo00000901000090000IVD00000000VDVLɔVD0VD309300VVDVO00LI50DI90V909DVOLI9VIVOLIVO99900VO99990V399VVOLIVO99VVOVVV39DVI3DDIOVD09LD00000Vop300D19llD1500DLlV3ovovvVDDDVOvpp0019DD9013039090D10vpDVDoDV10DV913VoDvpvvovpoVo9VOLllLD0090Lp0vovovVLOl0VovDovDo1vO0150DL1p0vo05000900vVvD00LLD09VVID1D0lIVDovoVD0150000VIVo0vOV9001V0o0VOL1VLD090V0IDVpDVDLID0009V0D09vOv39ɔVVLDD.LIVVɔpVVODVDooVOID0DVOvopp00DLIID.L200SIOVVDDIV9DIDDI9DVODIV9OVVD0999ID90000I9ID09009990LIVLID09VVI9V9p90DVGB/T43166—2023GACTTCGTAGCTGAGGCTTGGGGAACGGTCCGCTTAAGCAGTTCGCACATTTCATAGATATACGGCCCGATATTCAGGTCGCGGTGTTGGACCTGGCAGCTTGGGTCGTGTACAGGTTCACGCAGGCCCTGCCCGAAGGCGGGGAGGGCCAGTTGCTGATCGGCTCTGCAGGGGGGATCACAACCATCGTGGAACACAACGGCGAGCGAGAACGCCAACGGGAAGACGATAACCAGGCGAACCAACCGACCAGAATGACTTATAGACAGTAGGGTAAGTCGCGCGAGAGTGGTTACCGACGCCG