目的为了提高微量样本中miRNA的检测通量和检测效率,本文建立了一种能够同时准确定量两种miRNA的双重实时荧光定量PCR(real time fluorogenic quantitative PCR)检测体系,并通过实际样本检测验证其应用于体液鉴别的效果。方法设计适用于...目的为了提高微量样本中miRNA的检测通量和检测效率,本文建立了一种能够同时准确定量两种miRNA的双重实时荧光定量PCR(real time fluorogenic quantitative PCR)检测体系,并通过实际样本检测验证其应用于体液鉴别的效果。方法设计适用于miRNA双重检验的相关引物及探针并优化实验体系组分,建立基于TaqMan技术的miRNA双重实时荧光定量PCR检测体系并验证其特异性、灵敏度和可重复性;使用此检测体系对58份不同体液样本中的miR-451a与miR-21-5p进行检测,并借助miR-451a与miR-21-5p的比值鉴定法评估该体系的体液鉴别能力;使用该检测体系样本数据确定的最佳截断值对模拟案件样本进行鉴别。结果优化的检测体系能够实现对血液与非血液、月经血与外周血的100%区分,同时可以实现对模拟案件样本的准确鉴别。结论该双重实时荧光定量PCR检测体系将时间和材料成本均缩短至原来的一半,为后续建立更多重的实时荧光定量PCR检测体系并应用于体液鉴别打下了基础。展开更多
目的 构建一种基于线粒体DNA和核DNA遗传标记的中华鲟及其潜在杂交种的鉴定方法。方法 根据美国国立生物技术信息中心(National Center for Biotechnology Information, NCBI)已公布的中华鲟cytb基因序列设计引物,并选用鲟鱼单拷贝核基...目的 构建一种基于线粒体DNA和核DNA遗传标记的中华鲟及其潜在杂交种的鉴定方法。方法 根据美国国立生物技术信息中心(National Center for Biotechnology Information, NCBI)已公布的中华鲟cytb基因序列设计引物,并选用鲟鱼单拷贝核基因fam43a扩增引物对中华鲟DNA进行扩增与测序,其中对fam43a基因扩增产物进行TA克隆测序。通过BLAST序列相似性检索确定样本来源,并基于邻接法构建鲟形目系统进化树。结果 中华鲟样本测序所得cytb和fam43a基因序列长度分别为728 bp和730 bp,且10个克隆群所测fam43a基因序列完全一致。基本局部比对搜索工具(basic local alignment search tool, BLAST)序列相似分析显示,所测cytb与fam43a基因序列均与GenBank数据库中的中华鲟相似性最高,相似性分别为99.86%和100%。系统进化分析显示,中华鲟样本所测得序列均与GenBank数据库中的中华鲟DNA序列聚类在一起,且与鲟形目其他物种位于不同分支。结论 联合使用线粒体DNA和核DNA遗传标记能够快速准确地鉴定中华鲟及其潜在杂交种,该方法能够满足法医日常检案的鉴定需求,为中华鲟的拯救和保护提供科学技术支持。展开更多
目的探讨短串联重复序列(short tandem repeat,STR)三带型基因座同一认定似然比(likelihood ratio,LR)的不同计算策略。方法通过忽略三带型基因座、基于人群中不同类型的观测值、基于三带型基因座形成机制推导公式三个模型,对三带型基...目的探讨短串联重复序列(short tandem repeat,STR)三带型基因座同一认定似然比(likelihood ratio,LR)的不同计算策略。方法通过忽略三带型基因座、基于人群中不同类型的观测值、基于三带型基因座形成机制推导公式三个模型,对三带型基因座似然比进行计算。结果得到三个模型六种策略的LR计算方法,并分析其保守性与局限性。结论根据三体综合征发生机制推导的三带型基因座LR计算公式,可得到较为准确且可用于司法鉴定的LR值。展开更多
The DNATyper^(TM)21 kit has good species specificity,typing accuracy,and locus amplification balance,which can be used for forensic genetic analysis such as individual identification and paternity testing.To improve t...The DNATyper^(TM)21 kit has good species specificity,typing accuracy,and locus amplification balance,which can be used for forensic genetic analysis such as individual identification and paternity testing.To improve the anti-inhibitor ability of this kit,the addition of bovine serum albumin(BSA),bovine thrombin(BT),FastStart Taq DNA Polymerase,TaKaRa Ex Taq Hot Start Version(Ex Taq HS),MyFiTM DNA Polymerase,and Klentaq DNA Polymerase(Klentaq)as PCR enhancers to the PCR reaction system was explored in the presence of different concentrations of inhibitors,such as indigo,humic acid(HA),hemoglobin,heme,and melanin.The results revealed that BSA exhibited high efficiency in improving the detection rate of STR loci;BT only helped to overcome the typing inhibition caused by indigo,melanin,HA,and heme;Four types of DNA polymerase had different anti-inhibitor effects on different inhibitors,among which Ex Taq HS was more effective than the other three DNA polymerases,but displayed comparatively lower resistance to hemoglobin than BSA and BT.This study provides basic data for further optimization of the kit through comprehensive analysis and facilitates its application in daily forensic investigations.展开更多
为实现常见法医体液类型的快速鉴定,本研究挑选出10种具有体液表达特异性的mRNA及3种管家基因,并通过筛选一步法RT-PCR试剂盒、调整引物浓度、优化反应温度和时间,成功建立了一种可同时检测13种mRNA的一步法RT-PCR实验体系。使用该体系...为实现常见法医体液类型的快速鉴定,本研究挑选出10种具有体液表达特异性的mRNA及3种管家基因,并通过筛选一步法RT-PCR试剂盒、调整引物浓度、优化反应温度和时间,成功建立了一种可同时检测13种mRNA的一步法RT-PCR实验体系。使用该体系对144份样本(包括外周血、月经血、精液、唾液、阴道分泌液)进行检测并分析,多数目标基因特异性良好,检测时长在3 h以内,对外周血样本检测灵敏度可达0.01 ng RNA,可在混合样本中检测到混合成分的目标mRNA。虽然室温保存3~4年样本会有标记未检测到,但本实验体系具有操作简单、混合样本鉴别能力强、耗时短的优势,具有良好的实战应用前景。展开更多
The AGCU X Plus STR system is a newly developed multiplex PCR kit that detects 32 X-chromosomal STR loci simultaneously.These are DXS6807,DXS9895,linkage group 1(DXS10148,DXS10135,DXS8378),DXS9902,DXS6795,DXS6810,DXS1...The AGCU X Plus STR system is a newly developed multiplex PCR kit that detects 32 X-chromosomal STR loci simultaneously.These are DXS6807,DXS9895,linkage group 1(DXS10148,DXS10135,DXS8378),DXS9902,DXS6795,DXS6810,DXS10159,DXS10162,DXS10164,DXS7132,linkage group 2(DXS10079,DXS10074,DXS10075),DXS981,DXS6800,DXS6803,DXS6809,DXS6789,DXS7424,DXS101,DXS7133,GATA172D05,GATA165B12,linkage group 3(DXS10103,HPRTB,DXS10101),GATA31E08 and linkage group 4(DXS8377,DXS10134,DXS7423).A major advantage of this kit is that it takes into account linkage between loci,in addition to detecting more X-STR loci.In order to evaluate the forensic application of 32 X-STR fl uorescence amplifi cation system,PCR settings,sensitivity,species specifi city,stability,DNA mixtures,concordance,stutter,sizing precision,and population genetics investigation were evaluated according to the Scientific Working Group on DNA Analysis Methods(SWGDAM)developmental validation guidelines.The study showed that the genotyping results of each locus were signifi cantly accurate when the DNA template was at least 62.5 pg.Complete profi les were obtained for the 1∶1 and 1∶3 combinations.A total of 209 unrelated individuals from Southern Chinese Han community,consisting of 84 females and 125 males,were selected for population studies,and 285 allele profi les were detected from 32 X-STR loci.The polymorphism information content(PIC)ranged from 0.2721 in DXS6800,to 0.9105 in DXS10135,with an average of 0.6798.DXS10135(PIC=0.9105)was the most polymorphic locus,with discrimination power(DP)of 0.9164 and 0.9871 for the male and female.The cumulative PD_(F),PD_(M),MEC_(trio) and MEC_(duo) valu es were all greater than 0.999999999.There were 78 different DXS10103-HPRTB-DXS10101 haplotypes among the 125 males,and the haplotype diversity was 0.9810.There was no signifi cant difference in the cumulative PD_(F),PD_(M),MEC_(trio) and MEC_(duo) values whether considering linkage or not.In summary,the new X-STR multiplex typing system is effective and reliable,which can be useful in human genetic analysis and kinship testing as a potent complement to autosomal STR typing.展开更多
文摘目的为了提高微量样本中miRNA的检测通量和检测效率,本文建立了一种能够同时准确定量两种miRNA的双重实时荧光定量PCR(real time fluorogenic quantitative PCR)检测体系,并通过实际样本检测验证其应用于体液鉴别的效果。方法设计适用于miRNA双重检验的相关引物及探针并优化实验体系组分,建立基于TaqMan技术的miRNA双重实时荧光定量PCR检测体系并验证其特异性、灵敏度和可重复性;使用此检测体系对58份不同体液样本中的miR-451a与miR-21-5p进行检测,并借助miR-451a与miR-21-5p的比值鉴定法评估该体系的体液鉴别能力;使用该检测体系样本数据确定的最佳截断值对模拟案件样本进行鉴别。结果优化的检测体系能够实现对血液与非血液、月经血与外周血的100%区分,同时可以实现对模拟案件样本的准确鉴别。结论该双重实时荧光定量PCR检测体系将时间和材料成本均缩短至原来的一半,为后续建立更多重的实时荧光定量PCR检测体系并应用于体液鉴别打下了基础。
文摘目的探讨短串联重复序列(short tandem repeat,STR)三带型基因座同一认定似然比(likelihood ratio,LR)的不同计算策略。方法通过忽略三带型基因座、基于人群中不同类型的观测值、基于三带型基因座形成机制推导公式三个模型,对三带型基因座似然比进行计算。结果得到三个模型六种策略的LR计算方法,并分析其保守性与局限性。结论根据三体综合征发生机制推导的三带型基因座LR计算公式,可得到较为准确且可用于司法鉴定的LR值。
文摘The DNATyper^(TM)21 kit has good species specificity,typing accuracy,and locus amplification balance,which can be used for forensic genetic analysis such as individual identification and paternity testing.To improve the anti-inhibitor ability of this kit,the addition of bovine serum albumin(BSA),bovine thrombin(BT),FastStart Taq DNA Polymerase,TaKaRa Ex Taq Hot Start Version(Ex Taq HS),MyFiTM DNA Polymerase,and Klentaq DNA Polymerase(Klentaq)as PCR enhancers to the PCR reaction system was explored in the presence of different concentrations of inhibitors,such as indigo,humic acid(HA),hemoglobin,heme,and melanin.The results revealed that BSA exhibited high efficiency in improving the detection rate of STR loci;BT only helped to overcome the typing inhibition caused by indigo,melanin,HA,and heme;Four types of DNA polymerase had different anti-inhibitor effects on different inhibitors,among which Ex Taq HS was more effective than the other three DNA polymerases,but displayed comparatively lower resistance to hemoglobin than BSA and BT.This study provides basic data for further optimization of the kit through comprehensive analysis and facilitates its application in daily forensic investigations.
文摘为实现常见法医体液类型的快速鉴定,本研究挑选出10种具有体液表达特异性的mRNA及3种管家基因,并通过筛选一步法RT-PCR试剂盒、调整引物浓度、优化反应温度和时间,成功建立了一种可同时检测13种mRNA的一步法RT-PCR实验体系。使用该体系对144份样本(包括外周血、月经血、精液、唾液、阴道分泌液)进行检测并分析,多数目标基因特异性良好,检测时长在3 h以内,对外周血样本检测灵敏度可达0.01 ng RNA,可在混合样本中检测到混合成分的目标mRNA。虽然室温保存3~4年样本会有标记未检测到,但本实验体系具有操作简单、混合样本鉴别能力强、耗时短的优势,具有良好的实战应用前景。
文摘The AGCU X Plus STR system is a newly developed multiplex PCR kit that detects 32 X-chromosomal STR loci simultaneously.These are DXS6807,DXS9895,linkage group 1(DXS10148,DXS10135,DXS8378),DXS9902,DXS6795,DXS6810,DXS10159,DXS10162,DXS10164,DXS7132,linkage group 2(DXS10079,DXS10074,DXS10075),DXS981,DXS6800,DXS6803,DXS6809,DXS6789,DXS7424,DXS101,DXS7133,GATA172D05,GATA165B12,linkage group 3(DXS10103,HPRTB,DXS10101),GATA31E08 and linkage group 4(DXS8377,DXS10134,DXS7423).A major advantage of this kit is that it takes into account linkage between loci,in addition to detecting more X-STR loci.In order to evaluate the forensic application of 32 X-STR fl uorescence amplifi cation system,PCR settings,sensitivity,species specifi city,stability,DNA mixtures,concordance,stutter,sizing precision,and population genetics investigation were evaluated according to the Scientific Working Group on DNA Analysis Methods(SWGDAM)developmental validation guidelines.The study showed that the genotyping results of each locus were signifi cantly accurate when the DNA template was at least 62.5 pg.Complete profi les were obtained for the 1∶1 and 1∶3 combinations.A total of 209 unrelated individuals from Southern Chinese Han community,consisting of 84 females and 125 males,were selected for population studies,and 285 allele profi les were detected from 32 X-STR loci.The polymorphism information content(PIC)ranged from 0.2721 in DXS6800,to 0.9105 in DXS10135,with an average of 0.6798.DXS10135(PIC=0.9105)was the most polymorphic locus,with discrimination power(DP)of 0.9164 and 0.9871 for the male and female.The cumulative PD_(F),PD_(M),MEC_(trio) and MEC_(duo) valu es were all greater than 0.999999999.There were 78 different DXS10103-HPRTB-DXS10101 haplotypes among the 125 males,and the haplotype diversity was 0.9810.There was no signifi cant difference in the cumulative PD_(F),PD_(M),MEC_(trio) and MEC_(duo) values whether considering linkage or not.In summary,the new X-STR multiplex typing system is effective and reliable,which can be useful in human genetic analysis and kinship testing as a potent complement to autosomal STR typing.
