An STR Melt Curve Genotyping Assay for Forensic Analysis Employing an Intercalating Dye Probe FRET*

Abstract: The most common markers used in forensic genetics are short tandem repeats (STRs), the alleles of which are separated and analyzed by length using capillary electrophoresis (CE). In this work, proof of concept of a unique STR genotyping approach has been demonstrated using asymmetric PCR and a fluorescence resonance energy transfer (FRET)‐based hybridization analysis that combines fluorophore‐labeled allele‐specific probes and a DNA intercalating dye (dpFRET) in a melt match/mismatch analysis format. The system was successfully tested against both a simple (TPOX) and a complex (D3S1358) loci, demonstrated a preliminary detection limit of <10 genomic equivalents with no allelic dropout and mixture identification in both laboratory‐generated and clinical samples. With additional development, this approach has the potential to contribute to advancing the use of STR loci for forensic applications and related fields.     

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。     

线粒体DNA nt8584、nt8701单核苷酸多态性研究

目的建立一种快速、准确的线粒体DNA单核苷酸多态性位点检测方法。方法收集62份无关个体血液样本,应用荧光定量PCR技术研究线粒体DNA nt8584、nt8701位点在中国汉族人群中的多态性。结果 nt8584位点检测到10例nt8584A、52例nt8584G,nt8584A/G变异频率为16∶84;nt8701位点检测到27例nt8701A、35例nt8701G,nt8701A/G变异频率为44∶56。结论所建立的荧光定量PCR方法分型准确、耗时短,适用于法医DNA检验。     

LC—MS／MS法测定蓝色圆珠笔油墨中的碱性染料

目的建立蓝色圆珠笔油墨中碱性染料的LC—MS／MS方法,为蓝色圆珠笔油墨的种类鉴别提供方法。方法用二级质谱寻找并确定结晶紫、甲基紫、维多利亚蓝B、碱性紫14、碱性蓝7和罗丹明B等碱性染料的特征性母离子／子离子对。收集50种蓝色圆珠笔．划线后对其笔道用0．5mm直径打孔器取样,乙腈超声提取。液相分离采用WatersXBridgeC18柱。流动相为0．1％甲酸缓冲液（A）-乙腈（B）,梯度程序洗脱。结果4个点的取样量足以满足检测需要,采用相对峰面积的定量方法．结果重现性好．RSD％≤2．3％。应用该方法对50种蓝色圆珠笔油墨中的碱性染料进行检测,区分率为94．4％。结论所建LC—MS／MS方法定性准确,定量可靠,为蓝色圆珠笔油墨的种类鉴别提供了方法。     

Visualising latent DNA on tapes

This study reports a simple method for visualising and screening latent DNA on tapes using a Diamond™ dye (DD) staining process followed by visualisation using a portable fluorescence microscope. Ten types of tapes were tested, which include those used currently by forensic laboratories for tape-lifting. All ten types were tested for: 1) their auto-fluorescence, 2) properties when stained with DD using three different DD solutions, and 3) PCR inhibition through a direct STR amplification technique. No background fluorescence was noted viewing four types stained with 20 x DD diluted with 0.01% Triton-X. Clear tape (Sellotape®), DNA-free tape (Lovell Surgical Solutions) and brown packing tape (Packmate™) did not inhibit direct STR amplification, while the other six types showed the inhibition of the PCR. The three tapes were selected to assess their cellular material recovery efficiency by comparing the number of stained cells within an entire fingermark before and after tape-lifting. Tape-lifting was performed either once, twice or ten times. The DNA-free tape (Lovell) used in many forensic laboratories gave poor recovery compared to the clear tape (Sellotape®) and brown packing tape (Packmate™). This simple visualising technique allows the cell location to be recorded, and only the area of tape where cells are present to be removed for DNA typing. The process is a simple and effective triage procedure that reduces the processing of tape-lift samples where there are no cells present.     

Increased sensitivity for amplified STR alleles on capillary sequencers with BigDye XTerminator™

Free fluorescent dyes from PCR primers or amplification products can interfere with the interpretation of STR alleles in an electropherograph especially when the profiles have a low signal intensity. These artefacts can be removed by using a simple procedure based on BigDye^® XTerminator™. This procedure requires limited amounts of PCR product, allows to do several loadings on a capillary sequencer starting from the same purified PCR product and also increases the sensitivity for detection of less amplified loci.     

利用分散染料对血手印染色的研究

本文着重就分散染料对非渗透性客体上血手印的显现方法进行了科学详细的阐述,且突破原有传统的血手印显现法,开辟出用分散染料显现血手印的新途径,在大量实验的基础上,用各种分散染料以铝合金、PVC板、矽钢片、油漆木、玻璃等非渗透性客体上遗留的血手印为主要实验对象,对血手印包括血潜手印进行显现。通过在长波紫外灯下观察比较效果,最终发现荧光黄Ⅱ分散染料显现效果最佳,其显现的血手印荧光强度高,显出的纹线清晰、连贯性好,尤其加入适量的媒染剂后,手印与背景的反差更大,效果更加明显,是一种可靠的手印显现用染料。     

光谱成像法显现浅淡文字

光谱成像法,是近几年法庭科学运用的一重要方法。笔者以热敏字迹、印油文字、无碳复写字迹、针击字迹、纯蓝墨水及蓝黑墨水字迹色料形成的浅淡文字为研究对象,运用光谱成像技术做了系列实验,在普通白光照射下产生光谱影像,与传统可见荧光检验比较效果相当,且检验范围扩大至无荧光效果的色料。实验表明,光谱成像法是显现浅淡文字的一种全新的、有效的检验方法。     

布鲁氏菌血清抗体量子点荧光微球检测试纸条的研制

为研制一种现场快速筛查布鲁氏菌病的免疫层析试纸条,本研究以量子点荧光微球作为示踪物,共价偶联布鲁氏菌外膜蛋白OMP22,并将布鲁氏菌外膜蛋白OMP28和抗OMP22的单克隆抗体喷涂于硝酸纤维素膜上分别作为检测线和质控线,组装试纸条。结果显示,量子点荧光微球试纸条检测阳性血清的最大稀释度为1∶512,检测布鲁氏菌病阳性血清的敏感性为99%,特异性为98%,与虎红平板凝集试验(RBPT)的符合率为99%。且与结核病、蓝舌病、病毒性腹泻、口蹄疫及小反刍兽疫血清无交叉反应。上述结果表明量子点荧光微球免疫层析试纸条检测速度快、灵敏度高、特异性强且成本低,适用于布鲁氏菌病的现场筛查。     

10个STR基因座荧光复合扩增体系的研制

目的建立10个STR基因座荧光标记复合扩增体系,并评价其法医学应用价值。方法在北京、山西、广东汉族,辽宁满族、西藏藏族群体中调查STR基因座遗传多态性,筛选出9个具有高度多态性和法医应用价值的STR基因座及性别基因座。构建四色荧光素标记复合扩增体系,制备等位基因分型标准物,编制分析软件,并对体系的种属特异性、灵敏度、稳定性、混合样本等检测能力进行考察。结果建立的复合扩增体系遗传稳定好,累积非父排除率可达0.999 96,累积个体识别率可达0.999 999 999 999 3;与CODIS系统均不存在连锁遗传;各基因座间布局合理、无杂峰、扩增结果清晰易辨,并可实现检测分析自动化。体系种属特异性较好,灵敏度为0.1ng,稳定性好,混合样本检出范围在2∶8～8∶2之间。实际案例检材检测结果好。结论本文建立的复合扩增体系在法医学实践中有较好的应用价值。