单细胞检验技术用于混合上皮细胞的分离和检验

目的建立单细胞显微捕获联合低体积扩增技术,用于混合上皮细胞检材分离检验。方法取5名男性口腔上皮细胞拭子浸泡液30μL,分别滴加到5份含同一女性皮肤表皮细胞拭子上,制成5份混合上皮细胞样本为实验组,同时制备同样的5份样本为对照组。实验组样本采用显微捕获单个口腔上皮细胞,并使用低体积扩增技术进行扩增;对照组用M48纯化试剂盒提取DNA,Identifiler试剂盒复合扩增,扩增体系为10μL。所有产物均用ABI 3130遗传分析仪进行STR分型。结果 5份实验组样本均得到男性STR分型结果,5份对照组样本则均仅得到混合分型结果。将该方法应用于1例强奸杀人案例检验,取得了满意效果。结论单细胞显微捕获联合低体积扩增技术可用于混合上皮细胞样本的分离检验。     

单细胞分离荧光原位杂交法用于男女混合血DNA分型

目的采用单细胞分离荧光原位杂交法精确分离混合血样中男性和女性细胞并进行分型检验。方法收集男、女性血,按照男∶女为1∶5、1∶10、1∶20制备混合血样,加入0.075mol/L KCl 600μL,轻混、放置30min后加入150μL固定液离心留沉淀涂片,利用Vysis 30-161050试剂盒进行荧光原位杂交,并用PALM激光显微捕获系统分离出男、女性细胞,使用Identifiler试剂盒复合扩增并进行检测。结果捕获8个血细胞即可得到完整的DNA分型,且随着细胞数目的增多,检出率逐渐提高而等位基因丢失率逐渐降低。10个血细胞的检出率最高,为93.75%。5μL男性血液与本实验各比例女性血混合用本文方法检验均可获得男性分型。案例混合血斑经检验获得单一男性和女性分型。结论单细胞分离荧光原位杂交法可用于男女混合血样本中DNA分型检验。     

Reduced volume PCR amplification reactions using the AmpFlSTR Profiler Plus kit

The forensic community continues to seek improvements in DNA typing methods on aspects such as sensitivity and efficacy. Reducing the volume of the AmpFlSTR Profiler Plus reagents offered greater sensitivity and improved the chance of obtaining useful results for samples with very low quantities of DNA and multiple source samples. On the downside, amplifications initiated with less than 0.4 ng of DNA exhibited a twofold increase in the standard deviation of peak ratios. This research suggested a twofold approach to analyzing samples. For samples with greater than 0.25 ng of DNA, a 25 microL reaction is appropriate. Samples that did not demonstrate quantifiable results, or that have less than 0.25 ng, can be amplified by drying the sample directly in the PCR tube and amplifying in a 5 microL reaction. The analyst can expect at least limited results with as little as 0.03 ng of DNA in the 5 microL reaction.     

Forensic analysis of soils using single arbitrarily primed amplification and high throughput sequencing

Soil is a remarkably complex, diverse, ubiquitous, and easily transferred material which can reveal highly useful information to assist forensic investigations. In spite of its potential usefulness, the use of genetic soil analysis appears to be currently underestimated in forensic practice. Herein we report on the use of single arbitrarily primed amplification followed by high throughput sequencing of DNA fragments for the comparison of soil samples. The composition and functional attributes of soil microbial communities from three different locations were compared and shown to be different based on the metagenomic sequencing data obtained.     

Development and validation of the AmpFlSTR Yfiler PCR amplification kit: a male specific, single amplification 17 Y-STR multiplex system

In the past 5 years, there has been a substantial increase in the use of Y-short tandem repeat loci (Y-STRs) in forensic laboratories, especially in cases where typing autosomal STRs has met with limited success. The AmpFlSTR Yfiler PCR amplification kit simultaneously amplifies 17 Y-STR loci including the loci in the "European minimal haplotype" (DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, and DYS393), the Scientific Working Group on DNA Analysis Methods (SWGDAM) recommended Y-STR loci (DYS438 and DYS439), and the highly polymorphic loci DYS437, DYS448, DYS456, DYS458, Y GATA H4, and DYS635 (formerly known as Y GATA C4). The Yfiler kit was validated according to the FBI/National Standards and SWGDAM guidelines. Our results showed that full profiles are attainable with low levels of male DNA (below 125 pg) and that under optimized conditions, no detectable cross-reactive products were obtained on human female DNA, bacteria, and commonly encountered animal species. Additionally, we demonstrated the ability to detect male specific profiles in admixed male and female blood samples at a ratio of 1:1000.     

Droplet-based optical trapping for cell separation in mock forensic samples

Optical tweezers have a wide range of uses for mechanical manipulation of objects in the microscopic range. This includes both living and static cells in a variety of biomedical and research applications. Single-focus optical tweezers, formed by focusing a laser beam through a high numerical aperture immersion objective, create a significant force, which enables controlled transport of a variety of different cell types and morphologies in three dimensions. Optical tweezers have been previously reported to capture and separate spermatozoa from a reconstituted simulated postcoital sample. We report herein the development of a simplified, more efficient cell transfer protocol that can separate and isolate both spermatozoa as well as leukocytes, with similar efficiencies as those previously reported. The new cell transfer method was used to separate sperm cells from a reconstituted mixture of spermatozoa and vaginal epithelial cells, with complete STR profiles developed from 50 cells with little evidence of contribution from the female contributor to the mixture. This modified protocol was then used to separate 21 samples of enriched leukocytes, with trapped cells ranging from 5 to 22 cells. Complete STR profiles were developed from as few as 10 leukocytes. Thus, with minimal sample preparation and a short trapping time, this method has the potential to provide an alternative to traditional differential extraction methods for separation of sperm:nonsperm mixtures while also providing versatility for separation of cells with differing morphologies.     

Direct amplification of STRs from blood or buccal cell samples

Forensic databasing laboratories routinely analyze blood or buccal cell samples deposited on FTA^® paper. Prior to PCR amplification of the STRs, the FTA^® samples must undergo multi-step sample purification protocols to remove the PCR inhibitors present within the sample and from the FTA^® paper. The multi-step sample purification protocols are laborious, time-consuming and increase the potential for sample cross-contamination.To eliminate the need for DNA purification, we conducted studies to optimize the PCR buffer and thermal cycling parameters to allow for direct amplification of STRs from blood or buccal samples on FTA^® paper. We evaluated the effect of various factors on the DNA profile including: FTA^® disc size, blood sample load variation, and buffer formulation. The new STR assay enables the direct amplification of DNA from single source samples on FTA^® discs without sample purification. The new STR assay improves the workflow by eliminating tedious steps and minimizing sample handling. Furthermore, the new STR assay reduces cost by eliminating the need for purification reagents and expensive robots.     

Sex determination of human blood micro-stains and single hairs using specific DNA amplification with polymerase chain reaction

Amplification of Y chromosome specific DNA in vitro enables a rapid and reliable sex determination of human minute traces such as blood stains and hairs. In presence of male DNA a band of 154 bp is visualized by agarose gel electrophoresis after amplification, this band is lacking in case of female DNA alone. Amplification of a sex independent DNA locus (such as a fragment from the alcohol dehydrogenase gene) generates identical reaction products for both sexes. This shows that the absence of a band is not due to the lack of trace DNA. It is possible to perform this technique with as little as 0.5 microliters of blood or with a single hair.     

单个细胞DNA样本的制备及其在灵敏度分析中的应用研究

目的建立一种用于高精确灵敏度分析的微量DNA样本制备方法。方法用显微捕获技术制备以细胞为单位的DNA样本,并用于ProfilerPlus试剂盒及ABI310遗传分析仪的灵敏度测试。结果建立了单细胞捕获操作方案,成功制备了依次含1～11个细胞的一组DNA分析样本,并可准确用于ProfilerPlus誖试剂盒及ABI310遗传分析仪的灵敏度研究。结论基于显微操作技术的高精确微量DNA分析样本制备方法可准确用于分析技术方法的灵敏度研究     

Recovery of single source DNA profiles from mixtures by direct single cell subsampling and simplified micromanipulation

Deconvolution of forensic DNA mixtures into their individual component DNA (geno)types is of great investigative value, though often complex and difficult. Two-person mixtures comprising a major and minor contributor are often easily interpreted although, when the DNA ratio of the two individuals is approximately equal (~1:1), deconvolution and interpretation becomes much more difficult. To address this issue, a physical separation of individual-, two- or three- cell subsamples prior to autosomal STR analysis was performed using a simplified micromanipulation technique paired with a decreased reaction volume and increased cycle number PCR. Using this method, single and multiple buccal epithelial cells were collected from a 1:1 two-person mixture (i.e. from individual 'A' and 'B') and directly amplified, omitting standard DNA extraction and purification steps. Single cell subsamples resulted in partial single-source profiles for both contributors while, in accordance with expectations of a quasi-binomial sampling schema, two- and three-cell subsamples resulted in single source informative partial profiles of individual A and individual B as well as complete consensus profiles, and equally mixed 1:1 (2-cell subsamples) and 2:1 (3-cell subsamples) admixed profiles of individual A and B. This proof-of-concept approach shows promise in permitting the DNA deconvolution of mixed samples where the individual contributors are present in similar amounts that would otherwise be difficult to interpret, resulting in an increase in evidentiary value. The subsampling approach can be readily investigated for DNA casework applications without additional investment in costly, new equipment, requiring only a stereo microscope and a tungsten needle.     

龙胆紫染色法在显微捕获单细胞分离技术中的应用

目的建立用于显微捕获单细胞技术的龙胆紫染色法并评价其应用效果。方法制作20枚口腔拭子脱落细包悬液,配制0．05g／mL龙胆紫染液。取100μL口腔细胞悬液加入0．15、0．25、0．5、1．0、2．0μL染色液,考察最佳染色浓度;争别染色5、10、30、60min,考察最佳染色时间;用最佳条件染色后抓捕3个细胞,采用联合LV-PCR技术扩增并进行DNA分驯佥测,进行重复试验20次,并对同一检材未经染色的抓捕细胞进行检测,用于比对STR分型成功率。将龙胆紫染色法用于案例检材的口腔上皮细胞分离检验。结果1001μL细胞悬液加入0．5止0．05g／mL龙胆紫染液,染色5min,胞核呈紫工色,与胞浆对比明显;染色时间延长不影响染色效果及细胞分离检验结果。优化后的龙胆紫染色法对低体积扩增无归显抑制（P〉0．05）。应用此染色法于案例检材,胞核标识清晰,STR分型结果达同一认定标准。结论龙胆紫染色法刚于细胞核的发现,有助于提高显微捕获单细胞技术的检测效能。     

Preliminary trials of low volume AmpFlSTR Profiler Plus amplification using AmpliGrid (AG480F) slides

Low volume PCR using the AmpliGrid (480F) slide system can potentially enhance the generation of more complete profiles from trace samples, in addition to providing a more cost-effective alternative for typing standard samples. Based on our preliminary results, implementation will require a reasonable investment in optimisation and validation for the intended purpose.     

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.     

多重置换扩增技术及其法医学应用展望

多重置换扩增技术(multiple displacement amplification,MDA)是一种新型的全基因组扩增技术,具有操作简单、产物质量好、产物量稳定和扩增较均衡的优点,可以扩增低拷贝、混合、降解、含抑制物的DNA检材,为后续分析提供良好的高质量的扩增产物,在法医物证学领域具有很大的应用潜力,本文对该项技术的研究进展及应用前景进行综述。     

Genotyping for DQA1 and PM loci in urine using PCR-based amplification: effects of sample volume, storage temperature, preservatives, and aging on DNA extraction and typing.

Urine is often the sample of choice for drug screening in aviation/general forensic toxicology and in workplace drug testing. In some instances, the origin of the submitted samples may be challenged because of the medicolegal and socioeconomic consequences of a positive drug test. Methods for individualization of biological samples have reached a new boundary with the application of the polymerase chain reaction (PCR) in DNA profiling, but a successful characterization of the urine specimens depends on the quantity and quality of DNA present in the samples. Therefore, the present study investigated the influence of storage conditions, sample volume, concentration modes, extraction procedures, and chemical preservations on the quantity of DNA recovered, as well as the success rate of PCR-based genotyping for DQA1 and PM loci in urine. Urine specimens from male and female volunteers were divided and stored at various temperatures for up to 30 days. The results suggested that sample purification by dialfiltration, using 3000-100,000 molecular weight cut-off filters, did not enhance DNA recovery and typing rate as compared with simple centrifugation procedures. Extraction of urinary DNA by the organic method and by the resin method gave comparable typing results. Larger sample volume yielded a higher amount of DNA, but the typing rates were not affected for sample volumes between 1 and 5 ml. The quantifiable amounts of DNA present were found to be greater in female (14-200 ng/ml) than in male (4-60 ng/ml) samples and decreased with the elapsed time under both room temperature (RT) and frozen storage. Typing of the male samples also demonstrated that RT storage samples produced significantly higher success rates than that of frozen samples, while there was only marginal difference in the DNA typing rates among the conditions tested using female samples. Successful assignment of DQA1 + PM genotype was achieved for all samples of fresh urine, independent of gender, starting sample volume, or concentration method. Preservation by 0.25% sodium azide was acceptable for sample storage at 4 degrees C during a period of 30 days. For longer storage duration, freezing at -70 degrees C may be more appropriate. Thus, the applicability of the DQA1 + PM typing was clearly demonstrated for individualization of urine samples.     

Current good tissue practice for human cell, tissue, and cellular and tissue-based product establishments; inspection and enforcement. Final rule

The Food and Drug Administration (FDA) is requiring human cell, tissue, and cellular and tissue-based product (HCT/P) establishments to follow current good tissue practice (CGTP), which governs the methods used in, and the facilities and controls used for, the manufacture of HCT/Ps; recordkeeping; and the establishment of a quality program. The agency is also issuing new regulations pertaining to labeling, reporting, inspections, and enforcement that will apply to manufacturers of those HCT/Ps regulated solely under the authority of the Public Health Service Act (PHS Act), and not as drugs, devices, and/or biological products. The agency's actions are intended to improve protection of the public health while keeping regulatory burden to a minimum, which in turn would encourage significant innovation.     

免疫磁珠法与差异裂解法检验混合样本的效果观察

目的分析应用免疫磁珠分离技术和差异裂解法对混合样本的DNA检验效果,评价其在法医学应用中的价值。方法制备含有不同数量精细胞与阴道上皮细胞混合悬液和植绒混合斑拭子,分别采用MOSPD3抗体、SPAG8抗体免疫磁珠法及差异裂解法对样本进行检验,观察分析不同比例的混合细胞悬液,以及不同保存时间植绒混合斑拭子DNA分型的正确率。结果当混合细胞悬液内精细胞与阴道上皮细胞比例约1∶10时,免疫磁珠法的正确率明显高于差异裂解法(P0.01);当比例等于或高于1∶1时,两种方法正确率的差异无显著性意义(P0.05)。植绒拭子混合斑的免疫磁珠法正确率明显低于差异裂解法(P0.01);随混合斑保存时间的延长,差异裂解法正确率无明显下降(P0.05)。结论免疫磁珠法适用于新鲜混合样本中精细胞的分离检验,且在精细胞含量较低时优势更明显,差异裂解法较适合陈旧混合斑的分离检验。     

Ultraviolet illumination as an adjunctive aid in dental inspection

Tooth-colored resin fillings have become increasingly popular as restorative materials. Their presence in the dentition presents a challenge to the clinician and the forensic odontologist, as detection of the fillings can be difficult both visually and radiographically. As they necessarily form part of the unique dentition of an individual, recognition of the resins is important for forensic identification. Alternative light sources have been used with success in various fields of forensic science. In recent years small LED flashlights emitting at specific wavelengths in the ultraviolet light (UV) range have been developed. Their low cost, small size, and ready availability makes their use practical in both forensic dental inspection and clinical settings. UV inspection is of interest because enamel, dentin and dental materials all have differing fluorescent properties when illuminated by UV light. It was one goal of this research to quantitatively assess the fluorescence properties of modern restorative resins in order to predict their behavior during inspection using UV illumination. The second goal was to demonstrate practical use of UV in dental inspection with examples of how different materials fluoresce. Quantitative measurements were obtained for optical emission wavelength and intensity for 15 modern resins using a spectrophotometer. Results indicated that resin brands fluoresce at different wavelengths and with varying intensities. Practical use and comparison of the flashlights revealed that the most useful excitation wavelengths for resin detection were in the UVA range (365 and 380 nm). Porcelain restorations and composite resin fillings exhibited different responses to these two wavelengths and thus use of both is recommended for forensic dental inspection.     

论改革中国的出入境检查制度

中国现有的出入境检查制度存在不足之处，例如法律欠缺、行政诉讼程序有瑕疵、查控电脑软件有漏洞等。建议通过完善立法、改进电子化管理程度、革新出入境检验技术以及提高执法人员素质等举措来改革中国出入境检查制度，以便落实反恐维稳等保障国家安全的战略对策。     

全基因组扩增技术及其在法医遗传学中的应用前景

全基因组扩增技术是近年来发展起来的新型PCR技术,它提供了一种从微量基因组DNA获取大量遗传信息的途径,为法医处理微量检材提供了一个有用的工具。本文综述了全基因组技术及其种类和原理和其在法医遗传学方面的应用前景。