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2 ABI测序仪应用简介


ABI公司测序仪的应用
, Ph.D. Technical Product Specialist chenyd@appliedbiosystems.com


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DNA测序仪应用概况 基因突变快速扫描:SSCP 植物AFLP分析 细菌真菌鉴定:MicroSeq ID 甲

基化研究 BigDye XTerminator

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DNA测序仪应用概况

ABI遗传分析仪家族
使用同样的试剂和软件

310

3130 3130xl

3730 3730xl

1
4

4

16

48

96
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测序仪三大功能
DNA测序— 测定DNA片段的碱基序列 片段分析— 测定DNA片段的长度和颜色 SNP分型— 测定多态或点突变

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测序仪可以用来做什么?
DNA测序
医学测序 细菌、真菌鉴定 杂合子、突变检测

SNP研究
SNaPshot Multiplex SNPlex 定量PCR TaqMan探针杂交

片段分析
基于STR的法医鉴定:HID 基于STR的基因连锁定位:LMS 突变筛查 :SSCP 农牧业育种:AFLP

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未知序列测序

100多种基因组已经 完成测序,许多新基 因组计划正在进行中 许多基因有待发现 理解生物学功能 解码人类疾病的路径 和机制

Diagram from Celera Genomics 7

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线粒体DNA测序
Deleterious mutations in genecoding regions Genetic variation in populations Human evolution Disease association studies 500-1000 copies of mtDNA per cell allows easier extraction

MITOMAP: A Human Mitochondrial Genome Database. http://www.mitomap.org , 2004 Image provided by Molecular Probes photo gallery. http://www.probes.com/servlets/gallery/, 2004

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比较基因组学
Genetic conservation and variation Finding answers to disease mechanisms How closely related are mice and humans? How many genes are the same?
www.pathology.washington.edu – Mouse Karyotype ZooWeb - Karyotypes home page – Human Karyotype

人类

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医学测序
VariantSEQr? Resequencing System
A complete application solution for detecting variants in thousands of disease-related human genes.

SeqScape? Software v2.5
Allows automatic detection of heterozygotes

Mutation Profiling Insertions & Deletions Development of diagnostic / prognostic assays for disease
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杂合子检测:发现新突变

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细菌和真菌鉴定
MicroSeq? ID Analysis Software
MicroSeq? 500 16S Bacterial Identification Kit MicroSeq? D2 LSU Fungal Identification Kit

DNA Sequencing of the 16S ribosomal RNA gene Classification of Eubacteria, Yeasts,and Filamentous Fungi Sequence polymorphisms Bacterial category A biothreat agents Food pathogens Same day results
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SAGE? Analysis

基因组范围的基因表达图式分析 鉴定转录子(mRNA)

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基于测序的HLA配型
Donor / Recipient compatibility prior to tissue transplantation HLA typing products - distributed directly by Celera Diagnostics for research purposes only.
1-866-235-3723
http://www.celeradiagnostics.com/cdx/HLA_Typing

Referenced to GENBANK: HLA–C 6p21.3 major histocompatibility complex, class I, C. http://www.ncbi.nlm.nih.gov

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功能强大的片段分析
? 片段长度多态性
? AFLP和T-RFLP

? BAC指纹图谱 ? 相对荧光定量
? LOH和QMPSF

? 构象分析扫描突变
? SSCP和CSCE

? InVivoScribe B细胞和T细胞clonality ? TILLING
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相对荧光定量
细胞淋巴瘤(cell lymphomas) 及其他肿瘤中的等位基因丢失 染色体不平衡 部分和全部染色体丢失
LOH (Loss of Heterozygosity) P53 17p13.1

非遗传的in-vivo LOH

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寻找新的SNP位点
Mutation Profiling - SNP Discovery and Analysis

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SNP扫描
SNPlex? Genotyping System

SNaPShot? Multiplex System SNP Validation Linkage Mapping and Association Studies Pharmacogenomic Research

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SSCP突变快速扫描

SSCP
Amplify region of interest Heat denature in HiDi formamide and NaOH

A T A T A T

G C G C

Rapidly chill and electrophoresis under non denaturing conditions

G

C

WT Wild Type
20

rfu

MUT
mobility

Mutant
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SSCP Workflow
Blood Tissue Cells Bact. DNA

Sample Setup

Electrophoresis DNA Extraction Data Analysis Amplification Sequence suspected Post PCR Purification mutants

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SSCP data on ABI 3130 Series

WT WT + Mut1

WT + Mut 2

Mut 1

Mut 2

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植物AFLP?分析

AFLP技术原理

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AFLP工作流程
Perform AFLP assay on genomic DNA templates

Mix AFLP product with size standard and run on CE platform

Analyze data using GeneMapper software

Export/Print/Review final results (Electropherograms, Genotypes data in binary format
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AB公司AFLP相关产品
Kits

? Instruments
? 310 ? 3130 ? 3730

Plant Mapping Kit Small Genomes (50-500Mb) Regular Genomes (500-6000Mb) Microbial Fingerprinting Kit Microbial and Fungal Genomes

? Size Standard
? GS500ROX

? Software
? GeneMapper v4.0
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AFLP Data Generated on AB Systems
? 两个实例
? Experiment 1 - Maize genomic DNA templates ,Two parental samples (P1, P2) and first generation offspring (F1) from a cross between P1 and P2 ? Experiment 2 – Hedysarum (legume) samples isolated from different locations in Utah AB plant mapping kit ? Optimal primers for maize (but includes instructions for other plant systems) Size standards – GeneScan?500ROX? Electrophoresis ? Standard 36cm POP-7? Polymer Fragment Analysis run module for Applied Biosystems 3130 or 3730 DNA Analyzers (DyeSet F) Data Analysis ? New AFLP? software analysis method in GeneMapper ? Software v3.7

?

? ?

?

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Expt 1: Analysis of Maize AFLP? Kit Samples in GeneMapper? Software

F1
Polymorphic peak Common peak

P2

P1

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Results: Genotypes From AFLP Analysis of Maize Inbreds and a Hybrid
? Genotypes results will be automatically generated in a standard binary format ? Table can be printed or exported for further analysis

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Experiment 2: AFLP collaboration with U.S. customer
? Customer Collaboration with Dr. Steve Larson from Utah State
University

? Collect samples of native Hedysarum (type of legume) species
from various locations in Utah

? Isolate DNA from samples ? Perform Amplified Fragment Length Polymorphism (AFLP)
analysis on samples

? Perform phylogenetic analysis for Hedysarum 品种variety
identification

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Internal Use Only – Do Not Distribute

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Customer Research Project Goals
? Using native plant species for revegetation of disturbed lands in Utah
State and ultimately Western United States

Picture courtesy of Haley Fiske
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Internal Use Only – Do Not Distribute

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Genotypes from AFLP Analysis of Hedysarum Lines

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Hedysarum Variety Identification by AFLP Analysis
HB U48 01 17

Hedysarum boreale

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Hedysarum occcidentale
HO U5 01 15

HB U2 01 06 HB U4 01 17 HB U4 01 13 HB U4 01 18 HB U4 01 14 HB U4 01 15 HB U4 01 16 HB U48 01 02 HB U49 01 05 HB U49 01 04 HB U4 01 19 HB U2 01 15 HB U49 01 18 HB U49 01 13 HB U49 01 09 HB U49 01 06 HB U49 01 19 HB U49 01 10 HB U49 01 02 HB U49 01 12 HB U49 01 14 HB U49 01 08 HB U49 01 16 HB U49 01 03 HB U49 01 07 HB U49 01 15 HB U49 01 01 HB U49 01 17 HB U2 01 10 HB U2 01 12 HB U2 01 01 HB U2 01 19 HB U2 01 04 HB U2 01 03 HB U2 01 05 HB U2 01 07 HB U2 01 08 HB U2 01 16 HB U2 01 17 HB U2 01 02 HB U2 01 09 HB U2 01 11 HB U2 01 14 HB U2 01 18 HB U2 01 13 HB U2 01 20 HB U48 01 01 HB U48 01 20 HB U48 01 08 HB U48 01 14 HB U48 01 07 HB U48 01 13 HB U48 01 11 HB U48 01 18 HB U48 01 09 HB U48 01 10 HB U48 01 15 HB U48 01 16 HO U5 01 03 HO U5 01 02 HO U5 01 01 HO U5 01 17 HO U5 01 20 HO U5 01 10 HO U5 01 19 HO U5 01 09 HO U5 01 18 HO U5 01 11 HO U5 01 14 HO U5 01 08

Rabbit Gulch

Cuttoff

Wasatch

Echo Reservoir

Analysis Courtesy of Dr. Steve Larson, Utah State University

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MicroSeq? 细菌鉴定
500 bp试剂盒 全长基因试剂盒

FDA Draft Guidelines
对药厂生产管理中微生物检测的要求

Sterile Drug Products Produced by Aseptic Processing Current Good Manufacturing Practice - Draft Guidance August 2003 Pharmaceutical CGMPs
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分子生物学手段鉴定微生物
传统方法:培养法,形态学表型分类法 常导致无结果 无生长,无发酵 需要几天才出结果 生长条件和物理条件会影响结果 需要预先掌握分型知识 e.g. Gram stain

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MicroSeq? Microbial Identification Kit
A complete, integrated system for bacterial and fungal ID 样品制备试剂
PrepMan? Ultra reagent – fast boil & spin method

鉴定试剂盒
细菌 500、细菌全长基因 和 D2 真菌试剂盒

软件
Tools for assembly, editing and alignment of DNA, phylogenetic trees, and reporting

标准数据库
Validated sequences derived from bacterial and fungal type strains

仪器
Automated sequencing systems and thermal cycler IQ/OQ validation packages
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试剂盒组成
?
Universal PCR Master Mix ? Add genomic DNA ? Single amplification reaction ? 500 bp amplicon Universal Seq Master Mix ? Two sequencing reactions Positive and negative controls ? E. coli genomic DNA 50 identifications
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1 PCR module 1 Seq module

? ? ?
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细菌鉴定策略
forward

reverse

全长 PCR (1540 bp) 双向测序,互相确认
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MicroSeq? 工作流程
or

Sample from colony or pure culture克隆挑选或培养 PrepMan? Ultra DNA isolation kit DNA提取制备 MicroSeq PCR module (1 reaction) PCR扩增 < 30 minutes < 2 hours

MicroSeq Cycle sequencing module (2 reactions) 测序反应 2 hours DNA Sequencing 测序 Overnight

MicroSeq analysis software and rDNA database 软件分析,结果报告
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< 10 minutes

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样品制备特别简单
? AB公司新的 DNA 提取试剂:PrepMan? Ultra Reagent ? 适用于各种类型的样品 (e.g. foods, tissue, bacteria, fungi)
把样品加到200 ?L PrepMan? Ultra试 剂中

取5 ?L上清液做PCR

boil 100 °C 10 min vortex 20 sec centrifuge 2 min.
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MicroSeq? 真菌鉴定
真菌鉴定试剂盒

rDNA在真菌染色体上的排列

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测序目标是D2
D2:The largest single domain 200–500 bp amplicon, two primers ideal for high-throughput identification robust zero drop-out rate 1 pg to 1 ng DNA for identification

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强项
客观: 无需专家,鉴定方法可靠 重现: 无需特殊培养条件,准确鉴定 利于不同实验室间交流数据。 简单: 通用引物对,鉴定上千菌种 无需分型知识 快速: 一天内得到鉴定结果

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强项:MicroSeq? ID 软件
操作系统:兼容MicroSoft ? Windows ? 2000、Windows ? XP 自动数据分析 详细的报告:输出格式:PDF, HTML, TXT 经过验证的数据库 用户可根据需要扩充数据库 安全设置:不同用户级别设置、密码保护、记录追踪 符合 FDA实验室管理要求:21CFR Part 11 requirements:

MicroSeqID Software v1.0 Elelctronic Records Features MicroSeqID Software v2.0 Electronic Signatures Features
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强项:MicroSeq?数据库
细菌数据库 (500 及 全基因) 涵盖几乎全部芽孢杆菌属,分支杆菌属,葡 萄球菌属和棒状杆菌属(Coryneforms) 每年新增50-100条 真菌数据库

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methylSEQr Bisulfite Conversion Kit 甲基化分析 重亚硫酸钠转化试剂盒

Applied Biosystems Products for Each Step of the Workflow
Sample Prep

DNA Extraction

6100 Nucleic Acid PrepStation

(Bisulfite Sequencing)

Methylation Mapping

Template Prep

Bisulfite Conversion Primer Design PCR PCR Cleanup Sequencing Reaction Sequencing Cleanup Capillary Electrophoresis

methylSEQrTM Bisulfite Conversion Kit Methyl Primer Express? 9800 Fast PCR System PSI-Clone 96 Well Plate Kit BigDye? Terminator v1.1 CentriSepTM Spin Columns
ABI PRISM ? 310 Genetic Analyzer, AB 3130 & 3730 DNA Analyzers

Analysis

Sequencing

Data Analysis

SeqScapeTM Software1

1

Alternative freeware software BiQ can be used for analysis; http://biq-analyzer.bioinf.mpi-inf.mpg.de ? 2006 Applied Biosystems

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methylSEQr? Bisulfite Conversion Kit 重亚硫酸盐转化试剂盒
gDNA定量 用 methylSEQr? 变性缓 冲液将样品DNA变性 加入methylSEQr?转化 试剂50C作用15小时 用methylSEQr?纯化柱 纯化 ? Easy ? Robust ? Usable for all down-stream ..analysis methods

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重亚硫酸盐转化处理改变了基因序列
? 基因序列的化学性改变
? ? ? ? ? Forward & reverse strand of treated gDNA Generation of “new“ complementary strands at 1st PCR Cycle Increased occurence of T/T-stretches, A/A-stretches Change of molecular weight “Missing Base“ in unmethylated DNA
gDNA_FW 5‘CCCACATCGGGCCCCTTGGCAGGCTCAGGCGTTGGCTCCTCGGGGC gDNA_RV 3‘GGGTGTAGCCCGGGGAACCGTCCGAGTCCGCAACCGAGGAGCCCCG Methylated BIS gDNA_FW 5‘TTTATATCGGGTTTTTTGGTAGGTTTAGGCGTTGGTTTTTCGGGGT NEW Unmethylated RV 3‘AAATATAGCCCAAAAAACCATCCAAATCCGCAACCAAAAAGCCCCA

BIS gDNA_FW 5‘TTTATATTGGGTTTTTTGGTAGGTTTAGGTGTTGGTTTTTTGGGGT NEW RV 3‘AAATATAACCCAAAAAACCATCCAAATCCACAACCAAAAAACCCCA
? 2006 Applied Biosystems

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引物设计:Methyl Primer Express?

● Downloadable for FREE at www.appliedbiosystems.com/methylprimerexpress ● Compatible with all GA instruments
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试剂的选择

? Aggressive BIS treatment 重亚硫酸盐转化 ? 不完全转化 ? DNA 降解 ? 样品得率降低 ? 不当的样品处理将导致分析结果的差异 ? Homebrew vs. methylSEQr? Conversion Kit
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克隆?
? 采用哪种方法取决于研究的目的

? 克隆模板测序Quantitative results only by cloning
? Population of fragments segregated by cloning ? Sequencing & separation of single samples/clones ? At a given CpG x% of clones will show a C > x% Methylation

? PCR 产物的直接测序
? Pool of fragments is sequenced ? Per given CpG the result will be
– Methylated – Non-Methylated – Mixed

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克隆?
只有进行克隆分析才能得到定量结果 Quantitative results only by cloning

对PCR产物进行直接测序 ? 基因甲基化分型

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相对荧光定量
LOH QMPSF

LOH实验步骤
? 采集组织样本
? 健康组织 ? 肿瘤组织

? ? ? ?

提取DNA 使用标记的STR引物作PCR扩增 电泳 分析数据
? 比较正常与肿瘤组织样本的杂合子marker的峰高 ? 确定潜在的LOH候选者

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LOH实验材料
?试 剂
? ? ?仪 ? ? ? ? ?软 ?
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Microsatellite kits/primers Size standards 器 310 Genetic Analyzer 3100-Avant/3100 Genetic Analyzer\ 3130/3130xl Genetic Analyzer 3730/3730xl Genetic Analyzer 件 GeneMapper? v3.7 software or higher
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LOH的GeneMapper数据
Healthy

Tumor

LOH Candidate

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GeneMapper?软件的Report Manager
Report manager可以用来: ? 定制运算 – 求和、求平均值、求比值 ? 扫描LOH – 峰高比 ? 设置用户定义的LOH候选者标志阈值

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在Report Manager中确定LOH candidate

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QMPSF简介
? 短片段多重定量PCR
Quantitative Multiplex PCR of Short Fragments

? 用于检测病理样本中的重复和大片段缺失 ? 步骤:
? 多重PCR,如多个外显子,正常对照和病理组织 ? 使用GeneMapper v3.7或更高版本的下列功能 成对比较峰高或峰面积
? Report Manager ? Custom plot color overlay ? Dye scaling
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QMPSF数据举例
MLHI和MLH2基因的多重PCR

Individual 1

Individual 2

*From Charbonnier et al. Cancer Research 60:2760-2763
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BigDye XTerminator

BigDye? Xterminator? Purification Kit
? ? ? ? ? ? ? ?
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Simple and easy to use Effective dye-terminator and salt removal Reduction of overall purification costs Improved purification success rate Easy to implement >48-hr sample stability Direct Injection Automatable
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Current Methods Require Multiple Steps (EtOH ppt)

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Simple Protocol with BigDye? Xterminator ? Purification Kit
Add Vortex Spin Inject

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残留染料影响碱基计算的准确性(1-100 bp)

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Dye Blobs Affect Neighboring Lanes

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Ethanol Precipitation vs. BigDye? Xterminator Purification? Kit
Ethanol

BigDye? Xterminator Kit

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Samples in Water are Not Stable at RT for >24 hours

Fresh

24 hours

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Samples are Stable at RT >48 hours with BigDye? Xterminator?

Fresh

48 hours

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Benefits
Enhances overall productivity

Simplified workflow

Reduced purification time

vs ethanol precipitation and commercially available kits More useable high quality bases (Q20)

Efficient removal of dye blobs

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谢谢!


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