SYBR GreenI实时荧光PCR技术在人类DNA定量中的应用

目的建立SYBR Green I实时荧光定量PCR技术在法医物证检验中的应用。方法应用SYBR Green I实时荧光定量PCR技术对各种生物检材进行定量分析,并在此基础上进一步分析STR。结果得到了实验的各种生物检材的准确定量。结论SYBRSYBR Green I实时荧光定量PCR技术是一种高效且廉价的检测基因拷贝数的方法,具有法医学应用价值。     

实时RT-PCR检测大鼠死后管家基因mRNA的时序性降解

目的研究实时荧光定量RT—PCR方法检测死亡大鼠管家基因mRNA时序性降解的可行性,为死亡时间（Dostmortem interval,PMI）推断寻找新的研究手段。方法应用SYBR Green Ⅰ实时荧光定量RT—PCR技术．检测死后不同时间大鼠脑和脾中管家基因GAPDHmRNA及β—actinmRNA的水平,结果用循环阈值（简称Ct值）表示,分析死后经过时间与Ct值的线性关系,并建立死亡时间推断回归方程。结果GAPDH mRNA和β—actinmRNA的Ct值均与PMI之间存在显著的相关性。结论SYBR GreenⅠ实时荧光定量RT—PCR在定量分析mRNA降解的研究中是一个较理想的技术手段。选用管家基因作为PMI推断的研究对象,可在法医检案中消除其他基因因为个体差异带来的误差,更具实用性。Ct值作为动态监测机体死后不同时间点的客观指标．与死后不同时间点的线性关系良好,推断死后经过时间尤其是晚期死亡时间较为理想。     

实时DNA定量技术的应用研究

通过应用TaqMan探针法和SYBR Green荧光染料法对常见的法医生物学检材进行DNA定量,对实时荧光定量PCR技术用于法庭科学样品定量检测的适用性进行研究。实验结果表明,该技术在DNA定量及抑制因素评估等方面具有重要的法医学应用价值。     

TaqMan探针技术用于X-SNP位点的分型

目的建立一种基于TaqMan探针技术的快速、准确且经济的实时荧光PCR方法,用于检测X染色体上的单核苷酸多态性(single nucleotide polymorphism,SNP)。方法选择X染色体上的13个SNP位点(X-SNP),针对每个位点分别设计1对PCR引物和TaqMan探针,进行实时荧光PCR扩增,对X-SNP位点进行分型。结果13个位点均符合Hardy-Weinberg平衡;多态信息含量分布为0.3497～0.3750,杂合度为0.4537～0.5021。建立的方法能够用于X-SNP位点的基因分型,检测结果与DNA测序结果完全一致。结论基于TaqMan探针技术的等位基因特异的实时荧光PCR方法灵敏、简单、快速,可实现对X-SNP位点的分型检测;所选择的13个X-SNP位点具有高信息量,在法医遗传学中具有潜在的应用价值。     

正交设计在实时定量PCR检测条件优化中的应用

目的探索正交设计在实时定量PCR检测条件优化中的应用，找出扩增ICAM-1基因的最佳条件。方法建立大鼠皮肤损伤模型，采用正交设计实时定量PCR的各影响条件，提取损伤处皮肤组织总RNA反转录成cDNA，cDNA作为模板，通过特异性引物扩增ICAM-1基因。对扩增曲线和融解曲线制定评分标准，量化不同实验条件下的扩增结果并进行统计分析。结果退火温度、模板量和引物浓度直接影响扩增效果，但三因素间无相互作用。结果经统计学分析表明退火温度61℃、模板cDNA用量0．4μg和引物终浓度150μmol／L为实时定量PCR检测ICAM-1基因的最佳条件组合。结论正交设计是一种切实可行、快速经济的实时定量PCR检测条件优化方法。     

定量检测毒品的蛋白芯片的研发

目的利用蛋白芯片技术建立一种快速、高通量的毒品定量检测方法。方法采用硝酸纤维膜作为蛋白芯片的基片,将多种毒品和蛋白偶联物抗原包被在芯片基片上后封闭,不同毒品的单克隆抗体与之结合,再用荧光染料Cy5标记的二抗孵育,样本中如果含有毒品成分,将会与包被的毒品偶联物竞争性地与毒品的抗体相结合,导致最终的荧光信号发生改变,荧光信号的变化与样品中的毒品的浓度相关。通过荧光芯片检测仪CCD可以进行定量分析,最终判断样本中毒品的含量。结果对506例样本进行定性定量检测,与GC-MS仪器比较后总相关性在88%以上,芯片的检测灵敏度比胶体金方法提高10倍,其检测的特异性达到99%。结论蛋白芯片是一种有效、准确的高通量检测毒品含量的方法。     

同时鉴定9种肉源种属的复合检测体系的建立及其应用

目的建立一种可以同时鉴定猪、牛、羊、鸡、鸭、猫、狗、鼠和鲤鱼的多重PCR检测方法,并测试其技术性能指标,评估其在法医学或食品安全事件中的应用价值。方法根据上述9种动物的线粒体细胞色素b基因,利用其片段中种间特异性强的序列设计引物,采用毛细管电泳检测平台对各种属PCR扩增产物进行检测,依据扩增片段长度的差异与标记的荧光对初始模板的肉源种属进行鉴别;并通过扩增特异性、实际样本测试、混合样本检测灵敏度等指标评价该方法在实际法医学或食品安全案件中的应用价值。结果经过验证,建立的同时鉴定9种肉源种属的复合检测体系对其中任意一个种属的检测特异性均较高,且灵敏度较强,能够满足绝大多数法医学或食品安全案件中的检测要求。结论本研究建立的肉源种属复合检测体系可以在法医学未知种属样本的鉴定及食品安全肉类掺假等案件中提供帮助。     

荧光定量PCR技术研究线粒体DNA nt16519单核苷酸多态性

目的应用荧光定量PCR技术,建立mtDNA nt16519位点的PCR分型方法。方法应用Primer Express3．0软件,设计1对Taqman MGB探针和1埘扩增引物,在探针的5’端分别标记FAM和VIC报告荧光,应用7500荧光定量PCR和直接测序两种方法,分别榆测62份血样和18份毛发样本的mtDNA nt16519单核苷酸多态性,比较二种方法结果的一致性。结果62份血样和18份毛发样本均得到了可靠的mtDNA nt16519分型结果。结论建立的荧光定量PCR的分型方法操作简单、结果准确,适用于法医DNA检案。     

mRNA在体液斑鉴定研究中内参基因的选择与应用

利用mRNA分析技术鉴定生物检材中各种体液斑迹的组织属性来源是近年来法医物证学的一个重要进展。终点逆转录PCR和实时荧光定量PCR技术是法医学实验室常用的mRNA检测技术,为保证实验结果的准确性,研究者通常选用组成性表达的管家基因作为内参,对不同样本提取RNA进行质控和标准化,以正确评估目标mRNA在各样本中的表达量。本文聚焦近年来使用mRNA分析技术进行体液斑迹组织属性来源鉴定的研究报道,对这些工作中内参基因的选择与应用效果进行综述。     

Development of a real-time PCR assay to control the illegal trade of meat from protected capercaillie species (Tetrao urogallus)

A rapid and highly species-specific real-time polymerase chain reaction (PCR) assay has been developed for the detection of capercaillie DNA (Tetrao urogallus) in meat and meat mixtures. The method combines the use of capercaillie-specific primers, that amplify a 142bp fragment of the mitochondrial 12S rRNA gene, and a positive control primer pair that amplifies a 141bp fragment of the nuclear 18S rRNA gene from eukaryotic DNA. SYBR(?) Green dye or TaqMan(?) fluorogenic probes were used to monitor the amplification of the target genes. Results obtained with the use of TaqMan(?) probes as detection platform increased the specificity of the real-time PCR assay in comparison with the results obtained using SYBR(?) Green. The proposed real-time PCR assay represents a rapid and straightforward method for the accurate identification of capercaillie that could be used by law enforcement agencies as a tool for the control of poaching and illegal trade of meat from this protected species.     

Are these food products fraudulent? Rapid and novel triplex-direct PCR assay for meat identification

Unintentional contamination and fraudulent labeling of food, especially in halal-certified products, breach both religious and international laws. DNA-based methods are widely employed to identify trace amount of contaminants for law enforcement. Direct PCR has proved successful in the DNA analysis from degraded samples and PCR-inhibited samples, but it has never been applied to meat identification from food products. In this study, we aimed to develop a multiplex direct PCR assay for simultaneous identification of three commonly consumed meats without the need to extract DNA. Species-specific primers were designed from the mitochondrial DNA using the alignment of sequences available on GenBank. The assay was validated for its specificity, sensitivity, and usefulness in market sample analysis. The results showed that a highly specific and sensitive multiplex direct PCR assay was developed and provided the expected PCR fragment of approximately 100, 119, and 133 bp for pork (Sus scrofa), mutton (Ovis aries), and chicken (Gallus gallus), respectively. Thirty-nine market samples were tested and a small number of fraudulent labeling was detected. In conclusion, we developed a rapid and inexpensive test for three meat species. This cost- and time-saving assay could be easily adopted in the world food hubs, which are mostly third-world countries.     

Age estimation charts for a modern Australian population

The ivory industry is the single most serious threat to global elephant populations. A highly sensitive, species-specific real-time PCR assay has been developed to detect and quantify African elephant (Loxodonta africana), Asian elephant (Elephas maximus) and Woolly Mammoth (Mammuthus primigenius) mitochondrial DNA from highly processed samples involved in the international ivory trade. This assay is especially useful for highly processed samples where there are no distinguishing morphological features to identify the species of origin. Using species-specific Taqman(?) probes targeting a region of the mitochondrial cytochrome b gene, we developed an assay that can be used to positively identify samples containing elephant or Woolly mammoth DNA faster and more cost-effectively than traditional sequencing methods. Furthermore, this assay provides a diagnostic result based on probe hybridization that eliminates ambiguities associated with traditional DNA sequence protocols involving low template DNA. The real-time method is highly sensitive, producing accurate and reproducible results in samples with as few as 100 copies of template DNA. This protocol can be applied to the enforcement of the Convention on the International Trade of Endangered Species (CITES), when positive identification of species from illegally traded products is required by conservation officers in wildlife forensic cases.     

人与动物mtDNA细胞色素b基因的序列差异

目的 探讨人与动物之间mtDNA细胞色素b(Cyt-b)基因序列差异及其种属鉴定。方法 采用1对Cyt-b基因通用引物对人和19种动物共171例样本的mtDNA进行PCR扩增,琼脂糖凝胶检测扩增产物,ABI 377测序仪及荧光测序技术分析扩增产物的DNA序列。结果 所有样本均检测到1条358bp的扩增片段;任何两种动物扩增片段的序列都不相同,人与19种动物的序列差异在18.9％-30.0％,19种动物之间的序列差异在5.9％-32.9％。同种动物不同个体间只有人、驴及小白鼠存在变异,最多有4个碱基变异位点(1.3％),其它动物未发现种内变异。结论 人与不同种动物的Cyt-b基因序列存在差异,以此可区分不同种属的动物。     

Robust detection of cow and female buffalo DNA through Real-Time PCR assay

Cow, Bos taurus, and female buffalo, Bubalus bubalis, are considered sacred animals that are a part of rural livelihood in India. The purity of products from these bovine species has significant sentimental implications in the dairy and meat industry. Therefore, the mitochondrial DNA and the sex origin, targeting the X and Y chromosomes from these bovine species, were selected to design three multiplex real-time probe PCR assays: Hi-PCR® Cow Detection Kit (MBPCR184), Hi-PCR® Buffalo Detection Kit (MBPCR185) and Hi-PCR® Cattle Sex Determination Kit (MBPCR186). Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines were followed to perform different studies using reference control DNAs. An Internal Reagent Control (IRC) was part of every assay, thus ensuring a successful reaction. The assays were 100% specific, with no cross-amplification of the two bovine species. The amplification of the X chromosomal target was observed for male and female DNAs, whereas Y chromosome amplification was observed only for the male DNA. The assays were 100% specific to the target genes in these organisms with no non-specificity towards any other targets or organisms. The limit of detection for sex determination was 0.01 ng/µl, whereas the differential capability of the assay was 3 copies/µl and 30 copies/µl for Bos taurus and Bubalus bubalis, respectively. The assays were reproducible at 1 ng/µl genomic DNA with 95% CI. The assays are open and compatible with other brands of Real-Time PCR systems used in forensic labs. The experiments presented here verify that the developed real-time PCR assays are robust, produce reliable and reproducible results for detection and differentiation of Bos taurus and Bubalus bubalis and their sex even at low DNA concentrations.     

Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies

We explore different designs to estimate both nuclear and mitochondrial human DNA (mtDNA) content based on the detection of the 5' nuclease activity of the Taq DNA polymerase using fluorogenic probes and a real-time quantitative PCR detection system. Human mtDNA quantification was accomplished by monitoring the real-time progress of the PCR-amplification of two different fragment sizes (113 and 287 bp) within the hypervariable region I (HV1) of the mtDNA control region, using two fluorogenic probes to specifically determine the mtDNA copy of each fragment size category. This mtDNA real-time PCR design has been used to assess the mtDNA preservation (copy number and degradation state) of DNA samples retrieved from 500 to 1500 years old human remains that showed low copy number and highly degraded mtDNA. The quantification of nuclear DNA was achieved by real-time PCR of a segment of the X-Y homologous amelogenin (AMG) gene that allowed the simultaneous estimation of a Y-specific fragment (AMGY: 112 bp) and a X-specific fragment (AMGX: 106 bp) making possible not only haploid or diploid DNA quantitation but also sex determination. The AMG real-time PCR design has been used to quantify a set of 57 DNA samples from 4-5 years old forensic bone remains with improved sensitivity compared with the slot-blot hybridization method. The potential utility of this technology to improve the quality of some PCR-based forensic and ancient DNA studies (microsatellite typing and mtDNA sequencing) is discussed.     

Quantitation of human genomic DNA through amplification of the amelogenin locus

An alternate method for quantitation of human genomic DNA is presented. Quantitative template amplification technology (abbreviated "Q-TAT") estimates the quantity of human DNA present in an extract by comparing fluorescence in X and Y amplicons produced from unknowns with fluorescence in a standard curve amplified from known quantities of reference DNA. Q-TAT utilizes PCR and electrophoresis with fluorescent detection/quantitation, precluding the need for new instrumentation, methodology, or quality assurance associated with slot-blot or real-time PCR. In a comparison study incorporating shared samples, Q-TAT was found to be more sensitive than widely used slot-blot methods but somewhat less sensitive than real-time PCR. Among samples containing DNA concentrations ranging from 100 pg/microL to 2-4 ng/microL, Q-TAT produced DNA concentration estimates that agreed reasonably well with either Quantiblot or real-time PCR. Q-TAT was reproducible with a typical coincidence of variation of about 35%. Quantitation of human DNA in this study involved summing fluorescence in X and Y amplicons in unknowns and quantitation standards. However, analyzing fluorescence in X and Y amplicons individually could allow estimates of male and female DNA present in mixtures to be made. Moreover, since X and Y amplicons exhibit sizes of 210 and 216 bp, respectively, the integrity as well as the concentration of the genomic DNA template can be assessed. Q-TAT represents an alternate method useful for the quantitation of human genomic DNA prior to amplification of STR loci used for identity testing purposes. The method uses existing equipment and procedures in conjunction with a well-characterized DNA standard to produce concentration estimates for unknowns that reliably produce STR profiles suitable for analysis.