DNA数据库建设中批量样本直接扩增检验法的应用

目的研究批量样本直接扩增法在DNA数据库建设中的应用。方法打孔机打取血滤纸600份,剪取芝麻大小的口腔拭子300份,放入96孔扩增板,加入扩增试剂后直接扩增,并对其中200份血滤纸用磁珠法,100份口腔拭子用96孔板Chelex-100法,经DNA提取后扩增检验进行比较。结果血滤纸采用直接扩增法及磁珠法STR检测成功率均为100%;口腔拭子采用直接扩增法的成功率为95%,采用96孔板Chelex-100法的成功率为94%。结论血滤纸和口腔拭子通过直接扩增均能获得很好的DNA分型结果,该方法省时省力,可用于DNA数据库建设。     

Efficacy of Several Candidate Protein Biomarkers in the Differentiation of Vaginal from Buccal Epithelial Cells*

Abstract: Currently, there is no accurate method to differentiate vaginal epithelial cells from buccal epithelial cells in biological samples typically encountered in forensic casework. This study tested the expression of a selection of candidate proteins in buccal and vaginal epithelial cells. We investigated six candidate biomarkers, such as loricrin, vimentin, stratifin, cytokeratin 4, cytokeratin 13, small proline‐rich protein 2, and involucrin, using Western blot analysis on whole protein extracts and immunohistochemistry (IHC) on intact cells in an attempt to identify cell‐specific markers that would differentiate these cells by microscopy. Involucrin, loricrin, and stratifin showed differential expression during Western blot analysis and were carried through to IHC. Although proteins unique to vaginal epithelial cells and buccal epithelial cells were not identified from among the proteins tested, the increased expression levels of two proteins, loricrin and stratifin in vaginal cells, when compared to buccal cells, do provide encouraging results in the search for epithelial cell‐specific markers.     

Cytochemical detection of ABH antigens in human body fluids

The use of the peroxidase-anti-peroxidase (PAP) technique has been described previously for the detection of cellular antigens and in particular ABO antigens from tissue samples (Pedal and Hülle 1984; Pedal and Baedeker 1985; Pedal et al. 1985). In this survey, the PAP method has been employed to study the detection of ABO antigens in cells from body fluids of particular interest to forensic science, namely buccal cells and vaginal cells. Also tested, but in a limited number, were mixtures of body fluids and semen samples. No false reactions were obtained from buccal cells, all samples corresponding to the ABO blood type of the donor. Preliminary results from vaginal cells, vaginal/buccal cell mixtures, and semen were encouraging but must be treated with caution due to the limited number tested. Vaginal smears contaminated with semen showed varying degrees of nonspecificity.     

Determination of ABO antigens in fingernails using the APAAP (immunoalkaline phosphatase) technique

Fingernail specimens with adherent nail-bed were taken from autopsy material with blood groups A, AB, B and O. Frozen 4-5-microns sections were submerged and floated carefully during each working step. Portions of fingernails were contaminated with blood and buccal cells, respectively. Furthermore, fingernail fragments of 8 volunteers were embedded in a biocomponent adhesive according to Grieve and Kotowski (Forensic Sci. Soc., 26 29-34) (1986) and cut by the usual microtome technique. APAAP staining is a proper method for demonstrating blood group antigens in fingernails from groove to margin. Frozen sections as well as smallest specimen embedded in a suitable adhesive are applicable for staining procedures. Using freshly prepared artificial stains, blood group constellations of red blood cells and/or buccal cells adherent on the surface of fingernails may be distinguished from the nail matrix.     

Typing of mitochondrial DNA from isolated cells of the human buccal epithelium

The authors have developed a method for molecular-genetic analysis of DNA from isolated cells for the purpose of forensic medical diagnostics. The method is based on the use of the laser capture microdissection (LCM) technology in combination with typing of mitochondrial DNA. Optimization of the conditions for amplification of polymorphic mtDNA loci in preparations containing minimal amounts of the genetic material was accomplished at the initial stage of the work. To this effect, the two-round polymerase chain reaction was employed that allowed the amplified material to be accumulated in the amount sufficient for sequenation. At the next stages, the system thus obtained was tested on the cell model (using isolated cells of human buccal epithelium). It was shown that the proposed method is suitable for the analysis of specific mtDNA characteristics in a single human cell.     

Immunohistochemical staining as a potential method for the identification of vaginal epithelial cells in forensic casework

There is currently no accurate method to identify vaginal epithelial cells uniquely. This study aimed to use a cell extraction procedure compatible with routine forensic sampling methods, and to investigate the expression of cytokeratin (CK), estrogen receptor-alpha (ERalpha), and phosphodiesterase 5 (PDE5) in order to distinguish between skin, buccal, vaginal, and external penile epithelial cells. Seminal fluid samples were also examined. Epithelial cell samples were fixed in formalin, embedded in agarose, and processed using histological methods. Antigen-antibody reactions were detected using the DAKO Envision+ detection system. CK was present in all cells from all five sources confirming the origin of cells as epithelial. Both ERalpha and PDE5 positively labeled vaginal, buccal, and skin epithelial cells. Although an antigen unique to vaginal epithelial cells was not identified, we have described a cell extraction procedure for use in the immunohistochemical detection of a wide range of antigens, an approach compatible with forensic diagnostics.     

The determination of buccal epithelial cells in forensic medical cytological research]

The morphology of isolated cells of multi-layer squamous non-horny epithelium (buccal, vaginal, urethral, rectal), stained with 0.05% nigrosine solution, was under study, and the sizes of these cells and their nuclei were measured. Specific markers were detected in the buccal epitheliocytes, that permit their reliable differentiation from other epitheliocytes studied by the size of the cells and nuclei, by a characteristic structure of the cytoplasm, etc. The findings may be used for the diagnosis of the origin of isolated cells of the buccal mucosa.     

A novel histological technique for distinguishing between epithelial cells in forensic casework

There are a number of forensic cases in which the identification of the epithelial cell type from which DNA originated would provide important probative evidence. This study aimed to develop a technique using histological staining of fixed cells to distinguish between skin, buccal and vaginal epithelium. First, 11 different stains were screened on formalin-fixed, wax-embedded cells from five women. Samples were analysed qualitatively by examining staining patterns (colour) and morphology (absence or presence of nuclei). Three of the staining methods – Dane's, Csaba's and Ayoub-Shklar – were successful in distinguishing skin epithelial cells from buccal and vaginal. Second, cells were smeared directly onto slides, fixed with one of five fixatives and stained with one of the three stains mentioned above. Methanol fixation, coupled with the Dane's staining method, specific to keratin, was the only technique that distinguished between all three cell types. Skin cells stained magenta, red and orange and lacked nuclei; buccal cells stained predominantly orange–pink with red nuclei; while vaginal cells stained bright orange with orange nuclei and a blue extracellular hue. This staining pattern in vaginal cells was consistent in samples collected from 50 women aged between 18 and 67. Identification of cell type from unlabelled micrographs by 10 trained observers showed a mean success rate of 95%. The results of this study demonstrate that histological staining may provide forensic scientists with a technique for distinguishing between skin, buccal and vaginal epithelial cells and thus would enable more conclusive analyses when investigating sexual assault cases.     

Evaluation of Skin Surface as an Alternative Source of Reference DNA Samples: A Pilot Study

An acceptable area for collecting DNA reference sample is a part of the forensic DNA analysis development. The aim of this study was to evaluate skin surface cells (SSC) as an alternate source of reference DNA sample. From each volunteer (n = 10), six samples from skin surface areas (forearm and fingertips) and two traditional samples (blood and buccal cells) were collected. Genomic DNA was extracted and quantified then genotyped using standard techniques. The highest DNA concentration of SSC samples was collected using the tape/forearm method of collection (2.1 ng/μL). Cotton swabs moistened with ethanol yielded higher quantities of DNA than swabs moistened with salicylic acid, and it gave the highest percentage of full STR profiles (97%). This study supports the use of SSC as a noninvasive sampling technique and as a extremely useful source of DNA reference samples among certain cultures where the use of buccal swabs can be considered socially unacceptable.     

Sex determination from buccal mucosa and hair root by the combined treatment of quinacrine staining and the fluorescent Feulgen reaction using a single specimen

In order to determine sex using a single specimen, buccal mucosa and hair roots obtained from male and female individuals were used. The specimens were first stained with quinacrine for the detection of the Y-chromatin, and subsequently were stained by the fluorescent Feulgen reaction using acriflavine for the detection of the X-chromatin. In the male specimens, the frequency of fluorescent spots of quinacrine-positive bodies was high, whereas that of acriflavine-positive spots was low. On the other hand, in the female specimens, the frequency of quinacrine-positive spots was very low, while that of the acriflavine-positive spots was high. These specimens were air-dried and were allowed to stand at room temperature for periodical observations. The result was that sex difference was distinguishable for 4 months by the combined treatment method.     

Isolation of DNA from saliva of betel quid chewers using treated cards

Betel quid (BQ) chewing, a common tradition in tropical areas, often poses a problem during collection and DNA analysis of buccal samples from many indigenous communities for population genetic studies and in forensic analysis of chewed BQ residues. This study evaluated the use of FTA card, a chemically treated filter paper, in collecting buccal samples from long-term BQ users and subsequent PCR-based analysis using nine STR markers. A low overall success rate of amplification was observed in the samples extracted using a standard organic extraction procedure (7%) as compared with those prepared using the FTA card (89%). The presence of inhibitors in liquid DNA samples was verified when control DNA failed to amplify in the presence of an equal volume of liquid BQ samples. The use of the FTA card is more practical during field sampling than handling tubes containing buccal swabs.     

美国反垄断法"本身违法"与"合理法则"适用范围探讨

"本身违法"与"合理法则"是美国反垄断法中的一对重要法则,两者各有其相对独立的适用范围."本身违法"适用于价格固定、市场划分、联合抵制、搭售安排及转售价格维持等案件,"合理法则"适用于纵向非价格限制、联营、企业合并等案件.20世纪80年代,受芝加哥学派思想的影响,"合理法则"与"本身违法"的适用范围有所模糊.由于美国是一个多元社会,而"本身违法"与"合理法则"具有重要的协调反垄断法多元价值目标冲突之功能,所以,两者的适用范围仍清晰可辨.     

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。     

A New Approach to the Investigation of Sexual Offenses—Cytoskeleton Analysis Reveals the Origin of Cells Found on Forensic Swabs*

Abstract: There are forensic inquiries in which an identification of epithelial cell types would provide important probative evidence. In cancer diagnosis, this information is yielded by histological examination of cytokeratin (Ck). Therefore, we tested 19 antibodies against different Cks (Ck1, Ck2e, Ck4, Ck5‐6, Ck7, Ck8, Ck9, CK10, Ck13, Ck14, Ck15, Ck16, Ck17, Ck18, Ck19, Ck20, Ck903, PanCkAE1_3, and CAM5‐2) on histological sections of epidermis, buccal mucosa, vaginal mucosa, penis, urogenital tract, and rectum and could identify two antigens unique to buccal‐cell and vaginal‐cell (Ck4) and skin epithelial‐cell (Ck10) cytokeratin. Subsequently, we developed an immunocytological technique for distinguishing swabbed skin and mucosal cells up to at least 1 year after sampling. By the detection of the Ck4 and Ck10 mRNAs in biopsy and laser capture microdissection collected samples via quantitative real‐time polymerase chain reaction, we were able to confirm our immunological findings. Hence, this study offers techniques to discriminate between skin and mucosal cells (buccal and vaginal) in forensic casework.     

Application of RapidHITTM 200 System in Forensic MedicineCSCD

Objective: To validate the analysis capability of RapidHITTM 200 system for four kinds of routine forensic samples and the recyclable capability of template, template DNA and PCR products in the process of twice duplicate detection. Methods: The buccal swabs underwent the test twice by RapidHITTM 200 system, and the template DNA and PCR products that arose in the system were also tested for two times. After four kinds of routine forensic samples were detected by RapidHITTM 200 system, the follow- up tests of the template, template DNA and PCR products that arose in the system were performed. Results: The STR loci could be detected in the buccal swabs by the system for the first time. However, part of the STR loci lost during the second test. And the peak value obtained in the second test was significantly reduced than the one in the first time. The average STR loci detection rates of the template DNA and PCR products were both less than 50% in the second test, which were significantly reduced than that in the first test. In addition, the analysis capability of the system for the tissues and buccal swabs was better than that for the blood and cigarette butts. Compared with the first test, the STR loci detection rate of the tested items, template DNA and PCR products decreased with the numbers of tests. Conclusion: RapidHITTM 200 system is more effective in retesting buccal swabs than other samples, whereas the items, DNA template, PCR products obtained in the first and second time cannot be directly used for the further application and study of forensic medicine. © 2018 by the Editorial Department of Journal of Forensic Medicine.     

Determination of clofelin in blood by gas chromatography-mass spectrometry

Gas chromatography--mass spectrometry is proposed for measuring clofelin (clonidine) in cadaveric blood. The method includes liquid-liquid extraction of clonidine from the blood, derivatization with pentafluorobenzyl bromide, and subsequent purification of derivatization products before chromatographic analysis. The range of 0.5-50.0 ng/ml covers therapeutic and lethal concentrations of clonidine in the blood. The method was tried on expert material in Chelyabinsk Regional Bureau of Forensic Medical Expert Evaluations and can be used in forensic chemical and toxicological analysis.     

Evaluation of direct PCR for routine DNA profiling of non-decomposed deceased individuals

Forensic DNA profiling is a standard method used in the attempt to identify deceased individuals. In routine investigations, and if available, the preferred sample type is usually blood. However, this requires the invasive re-opening of the body, days or weeks after the autopsy, which is undesirable in resource-constrained mortuary settings. Motivated by the ease of sampling as well as reduced health and safety risks, this study aimed to establish the success rate of generating a full DNA profile on first attempt from buccal swab lysates using a direct PCR approach. Buccal swab samples were collected from 100 unidentified deceased males, and were subjected to direct DNA profiling with use of the Promega PowerPlex® Y23 Kit. At the time of sample collection, these individuals had been stored for between 1 and 887 days. This study shows that full DNA profiles were initially obtained from 73% of samples, which constitutes the first empirical data pertaining to first time success rates of direct PCR from post-mortem buccal lysates. Further investigation of partial and failed DNA profiles using real-time PCR showed that samples did not contain PCR inhibitors, DNA was not degraded, but DNA concentration was particularly low. Repeating DNA profiling with increased lysate input and extra PCR cycles yielded an additional six full DNA profiles, resulting in an overall success rate of 79%. Overall, DNA profile success rate was not associated with the duration of storage (p = 0.387). Lastly, massively parallel sequencing with the ForenSeq™ Signature DNA Prep kit provided more informative profiles for three additional samples. These results indicate that blood should therefore remain the sample of choice in a post-mortem setting, yet buccal lysates hold potential to be optimised further, which may ease the human identification workflow.     

基质辅助激光解吸飞行时间质谱分析生物样品中甲基苯丙胺

目的建立尿样和头发中甲基苯丙胺的基质辅助激光解吸飞行时间质谱（matrix-assisted laser desorption/ionization time of flight mass spectrometry,MALDI-TOF-MS）分析方法。方法尿样采用液液提取,头发经0.1mol/L盐酸水解后采用液液提取,以碳纳米管为基质应用MALDI-TOF-MS法检测。结果尿样中甲基苯丙胺的最低检测限（LOD）为0.5μg/mL,线线范围为线性范围为0.5～100μg/mL（R2=0.9970）;毛发中甲基苯丙胺的最低检测限（LOD）为0.4ng/mg,线性范围为0.4～60ng/mg（R2=0.9976）,对送检案例中尿样和头发检材进行检测,效果良好。结论本方法适用于尿样和头发中甲基苯丙胺的分析,与传统气相色谱质谱联用和液相色谱-质谱联用相比,分析速度更快,适合大批量样品同时分析。     

Internal Validation of RapidHIT®ID ACE Sample Cartridge and Assessment of the EXT Sample Cartridge*†

A new rapid DNA solution, the RapidHIT^®ID, can accommodate two different sample cartridges, ACE, for the analysis of a single swab and EXT, for the analysis of DNA extracts. An efficient internal validation designed for low‐throughput rapid DNA is described. An evaluation of the EXT sample cartridge is also described. Each cartridge generated profiles with sufficient data quality to meet CODIS eligibility in fewer than 120 min. The results exhibited 100% correlation when compared to conventional DNA typing methods. Precision, reproducibility, stochastic, mixture, and contamination experiments produced expected results. Sensitivity of the ACE sample cartridge was acceptable for buccal swab analysis. The sensitivity of the EXT sample cartridge is discussed. The ACE validation and the EXT evaluation utilized a minimalist, cost‐saving, efficient design to generate a validated RapidHIT^®ID instrument capable of producing genetic profiles from both extracted forensic DNA samples and buccal swab samples within 120 min.     

The development of a method for FISH identification of forensically relevant body fluids

Messenger RNA profiling is a useful confirmatory test for body fluid identification, but there are limitations to this method including sensitivity and the difficulty in linking body fluids with the corresponding DNA profile in mixed samples. We have developed a method for RNA suspension fluorescence in situ hybridisation (RNA S-FISH) for forensic-type samples using locked nucleic acid (LNA) probes. Vaginal and buccal epithelial cells have been the primary focus, with some preliminary work performed on leucocytes and seminal round cells. We have designed probes for the KRT10 messenger RNA and the micro RNA 891a. Using these probes, we have optimised a RNA S-FISH methodology and have successfully visualised these cell types using fluorescent microscopy.