9色荧光STR复合扩增体系的构建及验证  

Development and Validation of 9-color Fluorenscent Multiplex STR Amplification System

作　　者：李晨佳 张哲 莫晓婷 常海龙[3] 袁丽萍 孙敬 张广峰 尚蕾 宋文 李万水 叶健 白雪 LI Chenjia;ZHANG Zhe;MO Xiaoting;CHANG Hailong;YUAN Liping;SUN Jing;ZHANG Guangfeng;SHANG Lei;SONG Wen;LI Wanshui;YE Jian;BAI Xue(College of Forensic Medicine,Hebei Medical University,Shijiazhuang 050017,China;Institute of Forensic Science,Ministry of Public Security,Beijing 100038,China;First Research Institute of Ministry of Public Security,Beijing 100044,China)

机构地区：[1]河北医科大学法医学院,石家庄050017 [2]公安部鉴定中心,北京100038 [3]公安部第一研究所,北京100044

出　　处：《刑事技术》2023年第2期153-160,共8页Forensic Science and Technology

基　　金：国家重点研发计划(2021YFC3300104);中央级公益性科研院所基本科研业务费专项资金项目(2020JB006)

摘　　要：构建一套包含39个常染色体基因座及Amelogenin性别基因的9色荧光STR复合扩增体系,并验证其在微量与降解检材中的应用效果。根据中国人群核心基因座推荐标准及实际办案需要,筛选出合适的候选基因座,设计引物并以9种荧光染料对其进行标记,构建复合扩增体系并优化;对该体系进行灵敏度、种属特异性、稳定性等确证实验,并通过检验案件样本做进一步评估。结果表明:一套包含39个常染色体STR基因座及Amelogenin性别基因座(其中28个基因座<300 bp)的9色荧光复合扩增体系构建成功。各基因座均衡性良好,灵敏度达0.125 ng,对常见PCR抑制剂具备一定的耐受能力。两性混合样本最低检测比例为1:4;适用于不同类型案件样本检测,且与市场上其他同类试剂盒比对分型结果一致,种属特异性较好。所构建体系首次采用9色荧光标记技术制成复合扩增试剂,一次性检出基因座数量多,系统效能高;该体系构成以miniSTR为主,对于微量降解检材具有较高的实际应用价值。More STR-locus enclosure is beneficial and favorable for forensic genetics to select an STR testing kit.Thus,such a purpose-oriented STR multiplex amplification system was here to establish with a combination of 39 autosomal STR loci and the Amelogenin dyed by 9-color fluorescent substances attaching to their primers,along with its forensic value being validated about the practical application including the DNA samples difficult to handle.Based on the recommended criteria for core locus/loci among Chinese populations and actual case requirements,the candidate STR loci were screened over their eligibility,together with the primers being designed.Nine-color fluorescent dyes were used to label the relevant primers.Then,purpose-different various PCR reactions and optimization process were performed to have the STR multiplex amplification system constructed.Sequentially,validation experiments were carried out into the newly-constructed system to evaluate its sensitivity,species specificity,stability and other crucial indicators including the ability to profile the STR genotypes of the samples from real cases.Therefore,a 9-color fluorescent multiplex STR amplification system was successfuly constructed,containing 39 autosomal STR loci and the Amelogenin(28 loci<300 bp).The system was validated of being well-balanced with a sensitivity of 0.125 ng and tolerance to commonly-met PCR inhibitors,the minimum ratio of distinguishing two-gender mixture being 1׃4,and suitability for testing different types of case samples.The system also demonstrated its profiling STR-locus genotypes were truly consistent with those obtained from the commercial kits of same kind.Moreover,the system was proved of fine species specificity.As presumably the first time till present of adopting 9-color fluorescent labeling design,the system has enclosed more loci into one single test,hence attaining large efficiency.Essentially,the system is mainly composed of miniSTR,therewith making it good at detection of trace and degraded DNA samples.

关 键 词：法医物证学 9色荧光 复合扩增 MINISTR 微量降解检材 

分 类 号：DF795.2[医药卫生—法医学]