马STR复合扩增体系的建立和应用

Establishment and Application of Multiplex Amplification System for Horse STR

本文为实现对马匹进行亲缘鉴定及个体识别,基于STR分型技术建立了一套包含20个STR基因座和1个性别识别位点的复合扩增体系,并对其进行性能评价以及实际样本扩增测试。应用荧光标记技术,选择HTG06、HTG07、COR022、CA425、HTG04、COR082、LEX54、COR069、AHT05、HMS03、HMS01、HMS06、HMS07、COR058、HTG10、ASB17、VHL20、HMS02、ASB02、LEX34、Amelogenin共21个遗传标记,进行引物设计、PCR复合扩增、CE平台测序,验证该体系的灵敏度、特异性及耐受性。对128份实际样本进行检测以及亲缘关系比对,并对其中的66份未知亲缘关系的样本基因做多态性统计。结果显示:该复合扩增体系检测128份马样本能够获得完整STR分型,在实际扩增中,无非特异峰产生,对于不同种属动物,例如牛、羊、猪、鸡、猫、狗、人DNA样本扩增时不出现特异性扩增峰;0.075 ng马标准品在10μL体系下仍可以获得完整分型;对于4种常见抑制剂的耐受性可以分别达到血红素200μmol/L、血红蛋白200μmol/L、腐殖酸100 ng/μL、EDTA 1.2 mmol/L。本研究建立的马STR复合扩增体系分型准确、性能优,可满足实验室对马匹亲权关系鉴定和个体识别的需求。

This article aims to construct and valuated a novel multiplex PCR system,which consists of 21 loci and is used to identify the parentage of horses.Applying fluorescent labeling technology,HTG06,HTG07,COR022,CA425,HTG04,COR082,LEX54,COR069,AHT05,HMS03,HMS01,HMS06,HMS07,COR058,HTG10,ASB17,VHL20,HMS02,ASB02 LEX34,Amelogenin,a total of 21 equine motifs,were subjected to primer design,PCR complex amplification and CE platform sequencing to verify the sensitivity,specificity and tolerance of the system.A total of 128 actual samples were tested and compared for genetic relationship.Among them,66 samples with unknown genetic relationship were analyzed by polymorphism analysis.The complex amplification system was able to detect 128 equine samples to obtain complete STR typing,and in the actual amplification,no non-specific peaks were generated,and no specific amplification peaks appeared for different species of animals,such as bovine,sheep,pig,chicken,cat,dog and human DNA samples.0.075 ng of equine standard could still obt ain complete typing under 10μL system.The tolerance of the four common inhibitors could reach 200μmo l/L for heme,200μmol/L for hemoglobin,100 ng/μL for humic acid and 1.2 mmol/L for EDTA,respectively.The STR multiplex amplification system established in this study is effective and reliable,which can meet the needs of laboratory for the identification of paternity of horses.