用特异兔抗人精蛋白抗体间接ELISA法检测人精斑

本文报告利用抗人精血清进一步精制而得的特异兔抗人精蛋白抗体进行间接ELISA试验检测人精斑的方法。经用61例各种常见体液斑检材及多人不同浓度的精液、唾液、乳汁、血液等体液稀释液进行测试,结果正确检出精斑的准确率达100％。对混合多人精液稀释液的最高检出稀释度为1/10000,非精斑检材均不出现阳性反应。该法的特异性完全适合精斑确证检验鉴定的需要,其灵敏度明显高于传统的精斑确证检验方法。本法操作简单,无需特殊仪器设备,使用的抗体来源方便,便于应用。     

5 μl PCR反应体系的建立

PCR技术最大的优点是使目的DNA片段成百上千万倍增加,尤其适合于微量检材检验,该技术已成为常规法医物证检验技术。实际检案中,多余的PCR扩增产物没有用处,更是为了节约成本,我们微量化了ProfilerPlus试剂盒反应体系至５μl,报道如下,供参考。１材料与方法１．１材料送检案件滤纸提取血样１０份,混合斑检材为花短内裤１条,卫生纸２份,阴道擦拭纱布２份,地上提取精斑１份,镜下均检见人精子。所用试剂、仪器为：ProfilerPlus试剂盒;９６００型PCR仪;３１０型遗传分析仪。１．２方法（１）用Chelex１００法提取１０份血斑…     

不同织物上血斑洗涤后DNA检验结果分析

目的分析评价织物上洗涤过的血斑DNA检验的效果。方法取棉、化纤和麻布块各25份,用40μL新鲜血均匀涂制成直径约1.5cm的圆形血斑,样本分为5组,分别在加有皂粉的洗衣机中洗涤1、3、10、30、60min;所有样本用IQ试剂盒提取DNA,用Identifiler PlusTM试剂盒扩增,并进行STR分型检测。结果各种载体血斑周边处检材中,仅棉布载体洗涤1min检材检出RFU200的峰,但等位基因丢失率可达50%以上,不能正确判型。各种载体血斑检材洗涤30min以内均可得到成功分型,但峰高以棉布最高、化纤最低,洗涤60min时仅棉布血斑检出率为100%,其他载体血斑均有明显的等位基因丢失及非特异性扩增,检出率低于60%。结论洗涤一定时间后的血迹仍具有DNA分型检验的价值,能否正确提取洗涤后的血迹检材是分型成功的关键,洗涤时间较长的检材判型应谨慎。     

一种基于FRET的PSA荧光探针的制备及法医学应用

目的设计合成靶向识别检测前列腺特异性抗原的有机小分子荧光探针,探索荧光探针在生物物证精液斑确证试验上的应用。方法基于荧光共振能量转移机理设计合成前列腺特异性抗原荧光探针,通过质谱、高效液相色谱法等对探针进行结构及纯度表征分析,采用荧光光谱法测试探针对前列腺特异性抗原的荧光响应,制作不同附着物的精液样品检材,利用倒置荧光显微镜观察记录荧光成像。结果成功设计合成了一种前列腺特异性抗原荧光探针,荧光光谱实验表明其对前列腺特异性抗原有较好响应,最低检测限为0.8μg/mL,不同附着物精液检材荧光成像实验进一步验证了荧光探针在精液斑确证试验上的应用。结论本研究设计合成了一种对前列腺特异性抗原响应灵敏的荧光探针,有望用于法医学领域精液斑的确证试验。     

生物检材中苯丙胺类兴奋剂和氯胺酮的LC-MS/MS分析

目的建立生物检材中苯丙胺类兴奋剂和氯胺酮的液相色谱-串联质谱(LC-MS/MS)分析方法。方法生物检材包括血液、尿液和毛发,采用稀释法和液液提取的前处理方法,应用两个不同的液相柱,优化LC-MS/MS分析方法,并考察了血液和尿液基质的离子抑制作用。结果同时分析苯丙胺和MDA,液相1在3m in内完成,液相2可用于确认分析或复杂基质分离。尿液稀释法检材用量少,前处理简便快速。毛发中苯丙胺类兴奋剂和氯胺酮的最低检测限(LOD)为0.005~0.05ng/mg。对送检案例检材产妇头发和胎毛进行苯丙胺类兴奋剂和氯胺酮的分析。结论本方法可用于生物检材中苯丙胺类兴奋剂和氯胺酮的同时分析,血、尿等生物检材的离子抑制作用是影响本方法灵敏度的主要原因。     

DNA Typer~(TM)15 plus直扩试剂盒在DNA数据库建设中的应用

目的测试DNA TyperTM15 plus直扩试剂盒的技术性能指标,评价其在DNA数据库建设中的应用价值。方法采用DNA TyperTM15 plus试剂盒,并使用IdentifilerTM和DNA TyperTM15试剂盒进行比较,设定不同体系和引物量、不同退火温度和循环次数以进行方法验证;设定不同模板量标准品、不同比例混合样本,取猪、狗、兔等动物的血液样品,血痕、骨骼、唾液斑等常见检材样本以及不同建库样本,以验证试剂盒灵敏度、特异性、稳定性以及混合样本、常见检材及建库样本的检测能力。结果直扩试剂盒分型结果准确,重复性好,灵敏度可达0.125ng,不同批次间试剂检测结果稳定,对不同检材有很好的适应性。10μL扩增体系时FTA卡和加强型血液采集卡取样直径应为0.5mm,而血滤纸、血液采集卡样本和经典型血液采集卡取样直径应为1.0mm。结论 DNA TyperTM15 plus直扩试剂盒的性能可以满足DNA数据库建设及检案的需要,可在相关实验中选择使用。     

分光光度法检测硫化氢中毒血液中的硫化血红蛋白

目的研究生物检材中硫化氢的检验方法。方法用分光光度法测定生物检材(血液)中的硫化血红蛋白。结果硫化血红蛋白在波长612nm处有特征吸收,且其在此处的吸光度与硫化血红蛋白的浓度成正比。结论硫化氢中毒案件可通过分光光度法测定中毒者血液中硫化血红蛋白的浓度来确定中毒的程度。     

精-血混合斑检出单一STR分型3例

两人或两人以上个体的体液、分泌液混合形成的斑痕称为混合斑。包括精-阴混合斑、精-血混合斑、精-精混合斑及血-血混合斑等。精-血混合斑因与血斑在肉眼观察上无明显差别,有时会被办案单位作为血斑送检,检验人员如果不予注意,可能漏检。笔者在日常检案中遇到3例精-血混合斑,均被办案单位作为血迹要求检验,实验室检出单一STR分型,因与案件情况不符,     

PuriTyper~(TM)法医学纯化试剂盒的性能验证

目的验证PuriTyperTM纯化试剂盒各项性能指标和法医学应用价值。方法收集及制备抗凝血液、常见案件检材(唾液、烟头、精液、毛发、指甲、骨骼及组织块)、斑痕样本(血斑、唾液斑、精斑)以及模拟添加抑制剂和模仿自然环境中放置的血斑。采用PuriTyperTM纯化试剂盒提取纯化并进行DNA定量,IdentifilerTM复合扩增试剂盒扩增,产物经ABI 3130遗传分析仪进行检测,Genemapper软件分析结果,对该试剂盒灵敏度、稳定性、重复性、检材适应性进行测试。结果采用该试剂盒提取0.1～40μL血液分别获得0.042～26.45ng/μL的DNA。3种斑痕样本DNA产量平行试验结果稳定。不同类型检材重复检验所获IPC的CT平均值在27.60至28.03之间。常见案件检材所得分型与已知结果均一致。结论 PuriTyperTM纯化试剂盒能够满足法医DNA检验的要求,对法医学实践具有重要的应用价值。     

水浸和洗涤血样本的DNA检验

目的对经水作用的血样本DNA分型检验结果进行分析探讨。方法全血样本分为两组,水稀释组用水将全血样本稀释5、10、20、25、30倍后制作血斑;洗涤组分为纯水手洗、肥皂弱洗、肥皂强洗、84消毒液浸洗和洗衣粉机洗等5种洗涤方式。所有样本用IQ试剂盒提取DNA,Identifiler PlusTM试剂盒扩增,并进行分型检测。结果血液稀释组中心部位检材,均无等位基因丢失,除30倍稀释样本外,峰高均衡性均大于70%;外周部位检材出现2～10个等位基因丢失,峰高均衡性均小于50%。洗涤组中除84消毒液洗涤样本未检出DNA谱带外,其余均无等位基因丢失,而峰高及均衡性以手洗和肥皂弱洗样本更好。结论经水稀释或洗涤剂清洗的血样本,即使联苯胺预实验结果为阴性,选取合适的检验部位,仍可获得DNA分型。     

GC-MS determination of gamma-hydroxybutyric acid (GHB) in blood

A fast and efficient procedure has been developed for the analysis of total mercury in human tissues and blood using a hydride vapor generator system coupled to an atomic absorption spectrometer (HVG-AA). Tissue and blood samples were digested in a pressurized microwave decomposition system and the digest diluted prior to formation of free mercury vapor and analysis by atomic absorption. Recovery studies performed on 10 spiked/unspiked pairs of human liver and on 10 spiked/unspiked pairs of human blood samples yielded average recoveries of 99.7% (CV=0.4%) and 101.2% (CV=0.5%), respectively. The method detection limit for liver and blood was 50 microg Hg/kg and 12.5 microg Hg/l, respectively. The "normal" concentrations of mercury in human liver and blood are 33-490 microg Hg/kg and 0.6-59 microg Hg/l, respectively [1]. This method is able to determine mercury poisoning levels and may also be applied to detect mercury near the lower levels of these "normal" ranges, using the standard addition method approach.     

Raman spectroscopy of blood samples for forensic applications

We investigated Raman scattering from human blood as a function of parameters that are relevant for forensic field analysis, such as substrate, sample dilution, individual from which the sample originates, and age of the sample. Peaks characteristic of blood components and in particular the hemoglobin peaks were routinely detected when blood was deposited on substrates that are not strongly luminescent, such as plastic, metal utensils and dry wall. Raman scattering from blood proved quite sensitive and blood samples with a dilution up to 1:250 could be measured for an excitation power of ～2 mW measured at the sample plane. The sensitivity of Raman scattering to diluted blood allowed measurement using blood reconstituted from fabric substrates, thereby alleviating issues related to luminescence and scattering from the substrate. The dependence of Raman scattering on sample age and individual was also investigated. We found that the relative intensities of scattering peaks depended on sample age and history. For example, the relative intensity of oxyhemoglobin peaks decreases after blood has dried. Fresh blood drawn directly from a donor without intermediate storage exhibits also scattering peaks at 1155 and 1511 cm(-1) which disappear after drying. The origin of these peaks is under investigation. We noticed, however, that they were not found in blood that had been stored for longer than one week in EDTA containers before analysis, thus requiring the use of fresh blood for future studies and validation purposes. The relative intensity of scattering peaks was also found to be somewhat dependent on the donor and, for a same donor, on the day on which blood was drawn.     

Photoluminescence of blood by acidic hydrogen peroxide—A preliminary test

In this study, the authors found that treating blood with 1 M HCl and 2% (w/v) 5-sulfosalicylic acid (SSA) in 1% (v/v) hydrogen peroxide mixture can produce photoluminescence of blood. SSA was added as a blood fixer. The photoluminescence was induced by irradiation of a forensic light source at 505 nm, which was detected using a 550 nm barrier filter. In this experiment, various level of acid and hydrogen peroxide were tested to find the optimal formulation of reagents, spot tests were conducted with diluted blood to test the sensitivity of this reagent, and impressions in blood left on porous/nonporous surfaces were enhanced. The sensitivity of this solution was slightly lower than Bluestar and was similar to leucocrystal violet or leucomalachite green on both porous/non-porous surfaces. The photoluminescence of blood treated with this reagent has been observed over 2 months. Using this reagent, it was possible to observe fingermarks or footwear impressions in blood on a black porous/non-porous surface. Through this, it was found that using this reagent could enhance bloodstains regardless of the porosity or color of the surface.     

Detection of cocaine in blood stains]

Cocaine is rapidly degraded in blood samples, and its degradation was found to be highly dynamic in nature. The analysis of blood spots dried on filter paper may provide a method to minimize the break-down of cocaine and to largely preserve the analytical profile of the parent drug and its hydrolysis products at the time of sampling. The short term stability of cocaine in 100 microL blood spots prepared from unpreserved and preserved (sodium fluoride, 0.25%) blood samples was compared to the stability of the particular whole blood specimens stored in tubes at ambient temperature and at -20 degrees C. Due to dehydration, both the chemical and the enzymatic hydrolysis of cocaine and its products could be stopped in dried blood spots. More than 75% of the initial cocaine concentration could be detected in the blood spots, and the analytical profile was ensured for 17 days. Provided its practical suitability, the spot technology should offer a simple approach to detect actual impairment of motorists taken in police custody in the view of section 24a of the German traffic act as well as in cocaine associated criminal cases.     

Forensic Analysis of Human Autopsy Tissue for the Presence of Polydimethylsiloxane (Silicone) and Volatile Cyclic Siloxanes using Macro FT‐IR,FT‐IR Spectroscopic Imaging and Headspace GC‐MS

This study describes effective and straightforward primary and secondary methods for the detection of silicone in human autopsy tissue. The primary method is polydimethylsiloxane (PDMS) specific and employs either macro‐attenuated total reflection Fourier transform infrared (ATR‐FT‐IR) spectroscopy for samples with a high PDMS concentration (relative to that of the matrix) or micro‐FT‐IR spectroscopic imaging in a reflection/absorption modality for samples with a low PDMS concentration. Although the secondary method is not PDMS specific, it employs headspace gas chromatography with mass spectrometric detection (HS/GC‐MS) for the detection of low molecular weight volatile cyclic siloxanes (VCS), which are characteristic marker compounds for PDMS. Overall, the combined results from the primary and secondary analyses provide reliable evidence for the presence of silicone.     

红外光谱法用于安钠咖中咖啡因和苯甲酸钠快速定性和定量分析

目的 建立安钠咖样品中咖啡因和苯甲酸钠快速定性和定量分析的红外光谱方法.方法 采用高纯度咖啡因和苯甲酸钠混合制样的方法制备定性和定量建模样品,通过分析混合样品的红外光谱图,确定安钠咖样品中咖啡因和苯甲酸钠的特征吸收峰.采用偏最小二乘法(partial least squares,PLS)建立红外光谱定量模型.结果 通过...     

Forensic Identification of Blood in the Presence of Contaminations Using Raman Microspectroscopy Coupled with Advanced Statistics: Effect of Sand,Dust, and Soil

Body fluid traces recovered at crime scenes are among the most common and important types of forensic evidence. However, the ability to characterize a biological stain at a crime scene nondestructively has not yet been demonstrated. Here, we expand the Raman spectroscopic approach for the identification of dry traces of pure body fluids to address the problem of heterogeneous contamination, which can impair the performance of conventional methods. The concept of multidimensional Raman signatures was utilized for the identification of blood in dry traces contaminated with sand, dust, and soil. Multiple Raman spectra were acquired from the samples via automatic scanning, and the contribution of blood was evaluated through the fitting quality using spectroscopic signature components. The spatial mapping technique allowed for detection of “hot spots” dominated by blood contribution. The proposed method has great potential for blood identification in highly contaminated samples.     

Sex determination from plucked human hairs without epithelial root sheath

For sex determination, it is difficult to examine the sex chromatin in the hair root devoid of the epithelial root sheath because of melanin deposition in the hair cortex cells. In the present study, the possibility of determining sex was sought by observing the frequency of Y chromatin in the nuclei of hair cortex cells from human hair root devoid of epithelial root sheath. The male hair contained nuclei in each of which one brightly fluorescent spot was recognizable. The average frequency of the fluorescent spot in the hair cortex nuclei observed in 90 samples from 30 males was 59.4%; the maximum frequency was 74% and the minimum 37%. On the other hand, the fluorescent spots were rarely found in the nuclei of hair cortex cells from female hair. The frequency of occurrence was 5.3% on average, the maximum frequency being 12%, the minimum 0%. The frequency distribution of the male group and female group exhibited two discontinuous peaks. The frequency of the fluorescent spots can therefore be used as a criterion for determining sex. The fluorescent spot in dried male hair was shown to be detectable in a frequency of 20 – 40% 4 weeks after plucking, indicating that the nuclei of hair cortex cells can be subjected to the examination for at least this period if the hair has been kept in a dry condition.     

mRNA在体液斑迹鉴定与组织来源推断中的应用

体液斑迹鉴定及其组织来源推断一直是法医学研究的重要方向之一,其传统的检验方法存在诸如假阳性率高、检材易破坏等缺陷,亟需更高效的确证实验。高度分化的体细胞表达特异的m RNA分子,可推断其来源:外周血、月经血、精液、唾液、阴道分泌物、接触斑等,且灵敏度、特异度及保存时间相对较为理想,是未来用于体液斑迹鉴别的理想遗传标记。本文对m RNA在体液斑迹鉴定和组织来源推断方面的应用及其前景进行综述。     

线聚焦显微激光拉曼光谱技术区分激光打印墨粉

目的利用Renishaw InVia激光显微拉曼光谱仪对打印墨粉进行区分。方法用785nm激发波长,采取线聚焦模式,50倍物镜条件下,测定了惠普等8个品牌25种硒鼓型号的30份激光打印机打印墨迹样品的拉曼光谱。结果 30种样品墨迹的谱图进行了分析,根据拉曼的异同,可将30种样品分为5大类。结论线聚焦显微激光拉曼光谱可用于区分不同种类激光打印墨粉。