Assessing the performance of quantity and quality metrics using the QIAGEN Investigator® Quantiplex® pro RGQ kit

The Quantiplex® Pro RGQ kit quantifies DNA in a sample, supports the detection of mixtures and assesses the extent of DNA degradation based on relative ratios of amplified autosomal and male markers. Data show no significant difference in the accuracy and sensitivity of quantification between this and the Promega PowerQuant® System, both detecting the lowest amount of DNA tested, 4 pg. Laboratory controlled mixed male:female DNA samples together with mock sexual assault samples were quantified across a range of mixture ratios. Analysis software detected mixed DNA samples across all ratios for both quantification kits. Subsequent STR analysis using the Investigator® 24Plex QS Kit was able to corroborate mixture detection down to 1:25 male:female DNA ratios, past which point mixtures appeared identical to single-source female samples. Analysis software also detected laboratory degraded DNA samples, with data showing a positive trend between the Degradation Index (DI) and length of time of sonication. When used on ancient remains the assay was able to triage samples for further analysis, and STR profiles were concordant with DNA quantification results in all instances. STR analyses of laboratory-controlled sensitivity, mixture, and degradation studies supports the quality metric obtained from quantification. These data support the use of the Quantiplex® Pro RGQ kit for sample screening and quantification in forensic casework and ancient DNA studies.     

非洲猪瘟TaqMan实时荧光定量PCR检测方法的建立与应用

为建立检测非洲猪瘟病毒(African swine fever virus,ASFV)的快速诊断方法,本研究根据ASFV SY18株p17蛋白的编码基因D117L的保守序列设计并合成引物和探针,建立了基于ASFV p17蛋白的编码基因D117L的TaqMan荧光定量PCR检测方法,并验证其特异性、灵敏性和重复性。结果显示,本研究建立的TaqMan荧光定量PCR检测方法的C_t值与标准品在1×10^9~1×10^1copies/μL范围内呈良好的线性关系,相关系数为0.998,斜率为-3.192,检测下限为10 copies/μL,且与其他能引起相似症状的猪源病毒无交叉反应。重复性试验结果显示,组间与组内变异系数均小于1.920 7%,重复性好。此方法可用于ASF的早期诊断和ASFV快速检测。     

Informativeness of NGS Analysis for Vaginal Fluid Identification

The identification of vaginal fluids in forensic examinations plays an important role in crime scene reconstruction. Molecular detection of vaginal bacterial communities can lead to the correct discrimination of body fluids. These kinds of studies can be performed through multiplex real‐time PCR using primers for a specific selection of bacteria. The availability of next‐generation sequencing (NGS) protocols provided for the extension of the analysis to evaluate the prokaryotes present in specimens. In this study, DNA was extracted from 18 samples (vaginal, oral, fecal, yoghurt) and analyzed by real‐time PCR and NGS. The comparison between the two approaches has demonstrated that the information developed through NGS can augment the more conventional real‐time PCR detection of a few key bacterial species to provide a more probative result and the correct identification of vaginal fluid from samples that are more forensically challenged.     

链球菌猪主要致病种和型多重PCR检测方法的建立

根据GenBank中登录的链球菌属保守基因(EF-TU)、猪链球菌种保守基因(GDH)、C群马链球菌兽疫亚种保守基因(M-like)、猪链球菌型特异性基因(SS1型CPS1I、SS2型CPS2J、SS7型CPS7H和SS9型CPS9H)的序列设计合成7对引物,建立了一次性在属、种、型3个水平上检测链球菌猪主要致病种和型(C群马链球菌兽疫亚种和猪链球菌1、2、7、9型)的多重PCR方法。结果显示,该多重PCR在退火温度为61℃时可一次性扩增出7条符合预期大小、序列正确的基因条带;特异性试验和准确性试验结果均符合预期,无非特异性、交叉反应、假阳性、假阴性条带出现;对16株临床分离鉴定链球菌的检测结果与细菌学鉴定及单纯PCR鉴定的符合率均为100%;敏感性为0.08ng/μL;5次重复试验的结果也完全一致。结果表明,建立的多重PCR方法具有高度特异性、准确性、敏感性和稳定性,适用于猪场链球菌病的监控和流行病学快速诊断。     

牛羊赤羽病病毒环介导等温扩增检测方法的建立

为建立一种能够快速、简便地检测牛、羊赤羽病病毒的分子生物学方法,根据赤羽病病毒特异性S基因的6个特异区域设计了2对引物,首次建立了赤羽病病毒环介导等温扩增(LAMP)检测方法,对LAMP反应条件进行了优化,并将其与普通PCR方法进行比较。结果显示,LAMP检测方法的特异性好,只对赤羽病病毒进行扩增,且操作简便。本研究确定的最佳反应温度为63℃,核酸检测的最低检出限为1.08ng/μL,比普通PCR的灵敏度高2个数量级。该方法为赤羽病病毒的一种最新的快速、简便的检测方法,可用于出入境牛、羊赤羽病的快速检测。     

Quantification of human mitochondrial DNA using synthesized DNA standards

Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real-time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay's 105-base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust.     

Developmental validation of feline, bovine, equine, and cervid quantitative PCR assays

Accurate DNA quantification is essential for optimizing DNA testing and minimizing sample consumption. Real-time quantitative polymerase chain reaction (qPCR) assays have been published for human and canine nuclear DNA, and the need for quantifying other forensically important species was evident. Following the strategy employed for the canine qPCR assay, we developed individual assays to accurately quantify feline, bovine, equine, and cervid nuclear DNA. Each TaqMan-based assay incorporates a genus-specific probe targeting the Melanocortin-1 Receptor gene and includes a piece of synthetic DNA that acts as an internal PCR control for detecting inhibition. Developmental validations were carried out following the revised guidelines of the Scientific Working Group on DNA Analysis Methods with modifications necessary for validation of nonhuman qPCR assays. All assays demonstrated the specificity, sensitivity, stability, reproducibility, accuracy, and precision required for forensic casework. The application of these assays to animal forensic DNA analysis has both conserved laboratory resources and improved genotyping results.     

中国北方汉族人群FUT6基因3个SNPs遗传多态性

目的对中国北方汉族人群FUT6基因编码区序列特征及等位基因多态性进行调查。方法测序分析30例中国北方汉族人FUT6基因整个编码区序列并鉴定其单倍型,采用复合PCR与复合限制性内切酶结合的RFLP法分析FUT6基因rs778805（C370T）、G855A及rs61147939（C907G）3个SNPs遗传多态性;应用Haploview4.1软件进行相关统计分析。结果 30例测序样本中共检出8个SNPs和6种单倍型,149例中国北方汉族个体C370T、G855A、C907G基因型分布均符合Hardy-Weinberg平衡。杂合度分别为0.544、0.416、0.510;多态信息含量为0.375、0.372、0.367;个人识别能力为0.600、0.651、0.603;非父排除率为0.187、0.186、0.183。PCR-RFLPs检出13种基因型,单倍型变异度为0.646。Haploview4.1软件分析表明3个SNPs处于连锁不平衡状态。结论中国北方汉族人群FUT6基因编码区序列呈现出高度多态性;C370T、G855A、C907G位点多态性分布良好,但处于连锁不平衡状态。     

应用多种STR技术对基因嵌合体进行亲权鉴定分析

目的 查找嵌合基因的来源并进行父母和孩子的亲权鉴定.方法 采用Chelex-100法抽提基因组DNA,用复合扩增和荧光检测技术对STR、X-STR和Y-STR基因座进行分型.结果 父亲为XX,XY基因嵌合体,其能够提供给孩子必须的遗传基因.结论 父亲为被检孩子的生物学父亲.     

Real-time polymerase chain reaction quantification of canine DNA

The accurate quantification of target DNA is an important step in the short tandem repeat analysis of forensic biological samples. By utilizing quantification data to control the amount of template DNA in the polymerase chain reaction (PCR), forensic scientists can optimize testing and minimize the consumption of limited samples. The ability to identify and quantify target DNA in mixed-species samples is crucial when it may be overwhelmed by nontarget DNA, as in cases of dog attack. We evaluated two quantitative real-time PCR assays for dynamic range, species specificity, and inhibition by humic acid. While both assays proved to be highly sensitive and discriminating, the Melanocortin-1 Receptor (MC1R) gene Taqman assay had the advantages of a shorter run time, greater efficiency, and safer reagents. In its application to forensic casework, the MC1R assay has been advantageous for quantifying dog DNA in a variety of mixed-species samples and facilitating the successful profiling of individual dogs.