基于重组质粒制备STR分型阳性参照物

Preparation of positive reference material for STR genotyping based on recombinant plasmids

目的基于重组质粒制备可用于校准法医STR分型的阳性参照物。方法以常用阳性参照物9948人类基因组DNA STR分型为依据,基于重组质粒构建包含CSF1PO、D7S820、TH01等40个常染色体位点,DYS391、DYS522、DYS385a/b等22个Y染色体位点以及性别判定基因座Amelogenin的STR分型阳性参照物。将重组质粒定量、稀释后等比例混合,分别应用于DNATyper~?19、DNATyper~?24、DNATyper~?Y、Amp F?STR~?Identifiler~?Plus以及Power Plex~?18D System五种扩增试剂盒。结果阳性参照物中各重组质粒浓度为0.01pg/μL^0.001pg/μL;应用于Amp F?STR~?Identifiler~?Plus PCR扩增试剂盒,基于重组质粒制备的阳性参照物与人类基因组DNA扩增检测结果差异较小;将此阳性参照物分别应用于不同公司、不同STR基因座的四种STR扩增试剂盒,电泳检测图谱显示各基因座基因型完整,分型正确,峰高相当,基因座间均衡性良好。结论基于重组质粒制备STR分型阳性参照物,是一种可以替代细胞系制备阳性参照物的方法,具有一定的参考价值。基于此方法制备的阳性参照物可适用于市面上常用的STR检验试剂盒,普适性较强,对法医DNA分型检测有一定的实用价值。

Objective To prepare a new type of positive reference material for forensic STR genotyping.Methods This positive reference material was prepared with recombinant plasmids and employed the STR genotypesof 9948 human genomic DNA. The recombinant plasmids were diluted and mixed at equal proportions. Thispositive reference material covers 40 autosomal STR loci, 22 Y-STR loci and the sex-determining locus Amelogenin.To evaluate this positive reference material, it was applied to five different multiplex STR systems, which wereAmpFSTR IdentifilerPlus, DNATyper19, DNATyper24, DNATyperY and PowerPlex18D. Results The optimalplasmid concentration for STR genotyping was determined to be 0.01pg/gL-0.001pg/gL. The positive reference materialworked well in all five STR systems with all loci genotyped. All typing results were correct and the peak heights wereequivalent. Conclusion This positive reference material based on the recombinant plasmids works well with diverseSTR systems and exhibits significant value for forensic DNA analysis.