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.     

MDCK细胞感染犬细小病毒后的差异表达谱分析

通过了解MDCK细胞感染犬细小病毒(CPV)后基因表达水平的差异,研究病毒对细胞的致病作用以及细胞抵御病毒感染的机制。利用表达谱基因芯片技术,分析持续感染犬细小病毒的MDCK细胞基因表达水平的变化情况,并用Real-Time PCR技术加以验证。结果显示,获得了359个差异大于1.5倍的表达基因(P0.05),占总基因数的1.53%,其中193个上调表达(0.84%),166个下调表达(0.69%)。对差异基因进行GO功能聚类分析,小部分涉及免疫应答、生长周期调控、信号转导和蛋白酶活性,其他大部分基因功能未知。利用Real-Time PCR随机验证5个基因在持续感染CPV后的差异表达,其结果和芯片杂交的结果一致。表明建立了MDCK细胞持续感染CPV后的差异表达谱,初步了解到CPV对宿主细胞的制约作用,引起部分细胞增殖调控相关基因发生了表达下调,以及细胞对感染的积极应答反应,部分免疫反应基因、肽链内切酶活性基因等发生了上调表达,从而为探索病毒的致病机理和宿主的抗病毒途径提供了试验基础。     

延边地区牛瑟氏泰勒虫病的分子流行病学调查

为了解吉林省延边地区牛瑟氏泰勒虫病的流行情况,分别采用血涂片染色镜检法和PCR方法对采自延边地区的206份牛血液样本进行了牛瑟氏泰勒虫病的分子流行病学调查。结果显示,PCR检测的阳性率为62.6%,显著高于血涂片染色镜检法检测的阳性率(17.5%)。采用统计学分析方法对PCR检测的206份血液样本进行了不同地区、不同年龄、不同品种及饲养方式的比较;结果,不同地区之间和不同品种之间瑟氏泰勒虫感染率差异不显著(P>0.05);而不同年龄阶段之间和不同饲养方式之间牛瑟氏泰勒虫感染率差异显著(P<0.05)。结果表明,吉林省延边地区是牛瑟氏泰勒虫病的流行地区。     

宁波甬江水域夏冬季节溺死兔内脏硅藻16SrDNA的鉴定

目的采用PCR法检测夏季7月和冬季12月宁波甬江水域溺死家兔内脏组织中硅藻16SrDNA,评估其在夏季7月和冬季12月溺死鉴定中的应用价值。方法于夏季7月和冬季12月分别选取30只大白兔,总计60只大白兔,随机分为溺死组(n=10)、死后入水组(n=10)、空气栓塞组(n=10)。各组分别提取心、肝、肺、肾组织,应用PCR技术检测上述兔内脏硅藻16SrDNA。结果与死后入水组比较,夏季7月兔溺死组心、肝、肺、肾组织中硅藻16SrDNA检出率明显增高(P<0.05);而冬季12月溺死组仅心、肺组织中检见硅藻16SrDNA,与死后入水组比较无明显差异(P>0.05);夏季7月溺死兔组心、肝、肺、肾组织中硅藻16SrDNA检出率明显高于冬季季12月溺死兔组(P<0.05);夏季7月和冬季12月空气栓塞组心、肝、肺、肾组织中硅藻16SrDNA均未检出。结论宁波甬江水域夏季溺死兔中硅藻16SrDNA检出率高,PCR技术可用于溺死诊断;而冬季硅藻16SrDNA检出率低,运用该技术诊断溺死应慎重。     

30个中低突变Y-STR基因座复合扩增体系的建立

目的建立30个中低突变Y-STR基因座的复合扩增体系,并对其性能进行评估。方法采用六色荧光标记技术,选择30个中低突变率、中高多态性的Y-STR基因座自行设计引物,进行复合扩增和毛细管电泳检测。检测该体系的准确性、特异性、灵敏度和耐受性,并观察其在混合检材的分型情况。结果采用本体系,分型结果准确稳定、特异性好、耐受性强,灵敏度达0.0625ng,混合检材在1:4的比例下,可获准确分型。结论本研究建立的30个Y-STR基因座的复合扩增体系分型结果良好,对于中国人群的法医Y-STR数据库建设和群体遗传学研究具有重要意义。     

Application of direct PCR in forensic casework

Direct PCR is fast becoming a popular method in forensic science due to the advantages of saving time and money in the lab while increasing the probability of obtaining substantial results has a positive rippling effect. A laboratory is able to reduce the time spent on processing trace DNA samples, which can lead to investigators receiving important information in a timely manner that may not have been possible using standard methods. This study highlights the benefits of direct PCR in forensic casework by analysing trace and touch DNA on a range of substrates and exploring the loss of initial DNA due to extraction.     

Low-cost direct PCR for aged and processed wildlife sample analysis

Direct PCR has been used successfully in wildlife forensic DNA analysis from several types of biological samples using specialized, commercial direct PCR kits. This is attributed to the proprietary chemicals provided in the kits such as pre-PCR buffer and modified DNA polymerases. These reagents can be expensive, thereby limiting their widespread adoption in developing countries, where wildlife crimes are often encountered. We report on a study to evaluate the possibility of using low-cost direct PCR assay for degraded and processed wildlife sample analysis. Phire^® and Q5^® polymerases were used, due to their relatively low cost, for direct amplification of six aged and processed sample types (dried skin, 30-year old hair, muscle tissue, bone, trace blood mixed in vodka, and dried soft antler). The result indicated that Phire^® Hot Start II DNA polymerase and Q5^® DNA polymerase performed similarly to commercially available direct PCR kit. The low-cost amplification could efficiently identify species origin from all aged and processed samples. We observed a rate of more than 80% amplification success and high PCR product concentrations sufficient for further sequencing. The assay proved to be cost effective and robust; thus, we expect it to be adopted by wildlife forensic laboratories in developing countries.     

Multiplex DNA amplification and barcoding in a single reaction for 454 Roche sequencing: A comprehensive study on the control region of the mitochondrial genome

The analysis of the non-coding region of the mitochondrial genome using Sanger sequencing remains a laborious and time-consuming assay with too low resolution for the identification of low-frequency heteroplasmy or for mixture interpretation. In this study, an experimental design was tested in which the complete hypervariable region of the mitochondrial genome was sequenced using a novel barcoding strategy. The strategy involves a single-step multiplex nested PCR and we demonstrate its effectiveness by sequencing two multiplex reactions of two amplicons each covering the complete hypervariable region of the mitochondrial genome for 58 reference samples, 30 of which were analysed in triplicate, and 10 casework samples, each analysed in triplicate, on a 454 Roche DNA pyrosequencer with GS FLX chemistry using Multiplex Identifier (MID) primers to discriminate between samples. The generated reads for forensic (±3600 reads/MID) and reference samples (±466 reads/MID) allowed us to evaluate the accuracy in SNP calling and the variation in heteroplasmy and sequencing error rates in homopolymeric stretches between replicates.     

脱落毛发线粒体DNA HV1区序列测定的研究

目的　对脱落毛发线粒体DNAHV1区序列测定方法进行研究。方法　嵌合扩增结合末端荧光标记DNA测序。结果　对 2 0例脱落毛发进行分析获得了明确的测序结果 ,与来自同一个体的血液所测得的DNA序列进行比较 ,完全相同。结论　嵌合扩增在对脱落毛发进行线粒体DNA多变区序列分析中是一种有效的方法 ,在法医DNA检验中具有实用价值。     

青岛地区汉族人群13个STR基因座的频率分布及法医学应用

目的　调查青岛地区汉族人群无关个体的 13个STR基因座 (D3S135 8、VWA、FGA、D8S1179、D2 1S11、D18S5 1、D5S818、D13S317、D7S82 0、D16S5 39、TH0 1、TPOX、CSFIPO)的基因频率分布 ,研究其遗传多态性及其在法医学个体识别及亲子鉴定中的应用价值。　方法　用美国ABI - 310型遗传分析仪对ProfilerPlus和Cofiler两个系统的 13个STR基因座的复合扩增产物进行毛细管电泳及四色荧光自动分析检测 ,基因分型软件为GeneScanv3.1和Genotyperv2 .5 .2。　 结果　获得 13个STR基因座在青岛地区汉族人群的基因频率分布数据 ,13个STR基因座的PIC >0 .5 ,DP >0 .71,CCE =0 .999999,TDP值接近 1,TPm =1.2× 10 -14 ,家系调查符合孟德尔遗传规律。　结论　ProfilerPlus和Cofiler两个系统的 13个STR基因座在法医学个体识别及亲子鉴定中具有较高的应用价值。