Electrophoretic analysis of non-human primates hair keratin

Keratin characterization through electrophoretic techniques has been used for species identification in forensic science and in taxonomic studies. In the present work, protein components solubilized from hair of non-human primates were evaluated to investigate whether there is any species-specific pattern in an evolutionary perspective, by grossly comparing hair native keratins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Extracted hair keratins for all specimens were separated by SDS-PAGE into 2–3 polypeptide bands with apparent MW in the range 39–54 kDa and into 3–7 polypeptide bands with apparent MW in the range 10–35 kDa. With this technique it was possible to distinguish different suborders, different families of the same suborder, and, sometimes, different genera from the same family. On the contrary, it was not possible to distinguish different species of the same genus and different specimens of the same species. With IEF, extracted hair keratins were separated into about 30 polypeptide bands with pI values in the range of pH 3.9–7.7. IEF discriminates poorly between different samples. Only in specimens from Papio genus did we find an additional polypeptide band. In conclusion, we found that the differences between electrophoretic patterns are largest for animals that are not closely related while specimens of the same species have the same patterns.     

Big game species identification by deoxyribonucleic acid (DNA) probes.

Species identification is important in many big game forensic science cases but cannot always be accomplished because of the lack of adequate techniques. The authors have developed deoxyribonucleic acid (DNA) probes for elk, deer, and antelope by isolating highly repeated satellite sequences. These DNA probes distinguish among deer, elk, and antelope, although not between different species of deer. Because of the high number of sequence copies per genome, these probes are extremely sensitive, requiring less than 10 ng of total genomic DNA. The developmental protocol for these probes is relatively simple and is applicable to many other species.     

The isoelectric focusing of keratins in hair followed by silver staining

An isoelectric focusing method followed by silver staining has been developed for the study of keratins which is as effective as two-dimensional electrophoresis and fluorography for hair species identification. Hair from dogs, rabbits, horses, cows, guinea-pigs, donkeys, sheep and cats were successfully identified. Narrow pH ranges were used to observe heterogeneity in human hair. Although this heterogeneity may be affected by environmental conditions, it may be of use in criminalistics.     

The detection of sedatives in hair and nail samples using tandem LC-MS-MS

Drug screening methods were developed to detect alprazolam, clobazam, clonazepam, diazepam, midazolam, oxazepam, temazepam, triazolam, zopiclone, and selected metabolites in human hair and nail samples employing liquid-liquid extraction and tandem liquid chromatography-mass spectrometry (LC-MS-MS). Hair and nail samples were obtained from patients who had recently discontinued or were currently prescribed one or more of the targeted drugs. Prazepam was used as the internal standard for all compounds. Some components in the hair matrix gave the same transitions as some of the analytes but did not compromise the analyses because their retention times differed from those for the target compounds. The analytical run time was 8-10min. Results of the hair analysis of a DFSA victim are also presented.     

扩增Amelogenin基因用于生物检材种属鉴定

目的扩增Amelogenin基因测定血痕或组织的种属,以确定在法医学检验中的应用价值。方法收集猪、羊、马等10几种常见动物的血痕或肌肉组织,应用PCR扩增Amelogenin基因,PAGE电泳,银染后观察结果。结果哺乳动物猪、牛、狗、羊、马、驴动物血痕扩增产物为1条带,片段长度102bp。猕猴检见106bp和112bp两条带,与人血痕没有区别。兔、猫、鼠血痕未检出特异性片段。其它常见物种鳝鱼、青蛙、鸭、鹅、鸽、鹌鹑、麻雀等均未见扩增产物。结论扩增Amelogenin基因进行种属鉴定,方法简单,灵敏度高,可应用于法医检案。     

A comparison of the sensitivity of typing group-specific component (Gc) and the phosphoglucomutase (PGM1) locus in control and casework bloodstains by three isoelectric focusing methods

The performance of typing group-specific component (Gc) in bloodstains by two isoelectric focusing methods followed by its detection with silver staining has been compared with an established forensic system of typing phosphoglucomutase (PGM1) locus phenotypes by isoelectric focusing (IEF) in 1 mm gels. For Gc typing ultra-thin isoelectric focusing (UTIEF) gels and immobilized pH gradient (IPG) gels were used. Both laboratory prepared stains and casework stains were examined. The Gc UTIEF method is approximately eight times more sensitive than the existing PGM1 1 mm IEF method for control and casework stains. However, on average, a larger amount of stain was taken from casework stains than control stains for each typing system. A total of 53 casework stains were examined. Comparable success rates of 62% and 64% were obtained for typing Gc on UTIEF gels and PGM1 by 1 mm IEF, respectively. A success rate of 55% was obtained for typing Gc on IPGs. Bloodstains that were over 200 days old were successfully grouped by all three methods.     

基于12s rRNA的犀牛角制品DNA条形码鉴定分析

目的通过对几种涉案犀牛角制品的12s rRNA条形码序列比对分析,探析12s rRNA在犀牛角制品种属鉴定中的应用可行性。方法以3个案件中的涉案犀牛角制品为材料,采用改良的基因组DNA提取方法,PCR扩增DNA条形码片段12s rRNA。结果通过序列比对与分析,表明12s rRNA可将涉案犀牛角制品鉴定到种的水平。结论 DNA条形码12s rRNA可以作为一个新手段对形态上无法鉴别的犀牛角制品进行准确的种属鉴定,为案件的定性与量刑提供可靠的依据。     

Ribosomal ribonucleic acid (rRNA) gene typing for species identification.

Deoxyribonucleic acid (DNA) typing of ribosomal ribonucleic acid (rRNA) genes was performed with a polymerase chain reaction (PCR) assay for species identification. A variable region of the 28S ribosomal RNA gene was amplified with primers complementary to flanking sequences phylogenetically well conserved. The products of twelve animal DNAs (human, Japanese monkey, dog, cattle, pig, cat, rabbit, mouse, rat, chicken, frog, and fish) were separated by polyacrylamide gel electrophoresis, each revealing a few bands ranging from 150 to 100 base pairs. The band patterns obtained from each DNA sample differed in number and size, which indicates the applicability of the method to species identification. Samples containing either as little as 1 pg of DNA or degraded DNA of 0.2 to 0.5 kb in length were able to give detectable bands. Postmortem human tissue DNAs were tested as an example. They showed a pattern identical to the human control one, which was distinct from those of the other animals examined.     

Isoelectric focusing of human hair keratins: correlation with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns and effect of cosmetic treatments.

A new isoelectric focusing (IEF) technique in polyacrylamide gels with 6M urea and 1.5% Nonidet P40 has been developed to characterize human hair samples. The phenotypes demonstrated with this procedure has been correlated with the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) patterns described by other authors. The method described can be applied in the forensic science analysis of a single human hair. Using the same IEF technique we have studied the changes in electrophoretic patterns of cosmetically treated hair. The characteristics of the modifications observed and its utility in forensic science work are also discussed in this paper.     

The use of hybrid isoelectric focusing for the detection of polymorphic proteins in blood stains

alpha1-Antitrypsin (Pi), transferrin (Tf) and orosomucoid (ORM) were determined in bloodstain extracts by isoelectric focusing (IEF) with carrier ampholytes (CA) and also with a mixture of immobilines (HIEF). HIEF yields superior results from proteins typing in bloodstain extracts, since phenotypes are better distinguished and the bands are straighter and sharper. Also the sensitivity of HIEF is similar to IEF with CA.     

Nail analysis for the assessment of caffeine abuse in Northwest China: A population-based study

Caffeine is the most widely consumed psychoactive agent worldwide and has the potential for abuse, but studies monitoring caffeine abuse in China are scarce. This study aims to estimate the prevalence of caffeine abuse in northwest China and investigate the correlation between caffeine and other drugs in hair and nails using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Fingernail clippings were collected from 376 participants in northwest China to detect caffeine and 13 other illicit psychoactive drugs and their metabolites. Paired hair and nail samples were collected from 39 participants to investigate the correlation between caffeine and other drugs in hair and nails. The samples were decontaminated, pulverized, and extracted by a high-throughput nail sample preparation method and analyzed by UPLC-MS/MS. The results showed a risk of caffeine abuse in northwest China, with concentrations ranging from 0.43 to 10.6 ng/mg for healthy volunteers, 0.49–246 ng/mg for caffeine abusers, and 0.25–363 ng/mg for drug addicts in community rehabilitation centers. Caffeine was detected together with other illicit psychoactive drugs and their metabolites. Furthermore, positive detection correlations were found between hair and nail samples. This study provides a current perspective on caffeine abuse in northwest China and demonstrates the practical use of UPLC-MS/MS for the simultaneous detection of caffeine and 13 illicit psychoactive drugs and their metabolites in hair and nails. The results highlight the potential of nails as a supplementary matrix when hair samples are unavailable and emphasize the need for handling caffeine carefully given its potential for abuse.     

An improved method for subtyping Pi in old bloodstains.

Immunofixation procedures were used for detecting alpha-1 antitrypsin protease inhibitor (Pi) phenotypes in bloodstains. Neuraminidase elution of bloodstains, together with isoelectric focusing, immunofixation, and silver staining techniques, makes possible Pi subtyping in old bloodstains. No extra bands appear when the storage time is no longer than three months.     

ABO基因型检验及其法医学应用研究

目的采用ＰＣＲ技术对ＡＢＯ血型系统进行基因型检验。方法选择最佳扩增条件进行四引物复合扩增,用限制性内切酶ＫｐｎＩ和ＡｌｕＩ分别酶解扩增产物,电泳分离、银染显色法检验ＡＢＯ基因型。结果对２７０例血斑、２０例混合斑、２０根毛发（有毛囊）、１２份唾液斑等不同的生物检材进行了分型,与血清学方法检验结果相符。结论该方法能够应用于法医学的检验     

Analysis of morphine by RIA and HPLC in fingernail clippings obtained from heroin users

Heroin is abused around the world and is frequently reported as the cause of death in overdose cases. Analysis of morphine in hair has been used in the past in forensic toxicology to study the addiction history of heroin addicts. The purpose of the present study was to evaluate the usefulness of the nail as an analytical specimen in the identification and quantification of morphine in fingernail clippings of known heroin users. Fingernail clippings were obtained from 26 consenting patients of the Glasgow Drug Problem Service. At the time of sampling, the participants provided answers to a questionnaire regarding their drug use patterns. Samples were decontaminated by sonication in SDS, deionized water and methanol, and the methanolic washes were screened for analyte presence. The washed nail clippings were then hydrolyzed and extracted. RIA was used for the screening and HPLC for the confirmation of morphine. Positive RIA results were obtained with nail clippings from 25 of the 26 heroin users. The levels ranged from 0.06 to 4.69 ng/mg with a mean morphine concentration of 1.67 ng/mg. HPLC results were positive for 22 of the 26 nail samples. The mean morphine level by HPLC was 2.11 ng/mg with a range from 0.14 to 6.90 ng/mg. Based on these results, we suggest that nails have the potential of becoming a powerful alternative to hair for the detection of past heroin use in forensic cases.     

Analysis of microsatellite polymorphism in red deer, roe deer, and fallow deer -- possible employment in forensic applications

DNA microsatellites play a major role in population genetics, linkage mapping, and parentage studies of mammals. In addition, they may be used for forensic purposes, if an individual identification of a specific animal is necessary. Therefore, we tested a variety of microsatellite polymorphism derived from reindeer (Rangifer tarandus) by PCR and sequencing analysis for use in red deer (Cervus elaphus), roe deer (Capreolus capreolus) and fallow deer (Dama dama). Twelve of these microsatellites were selected for further analysis. In all these microsatellite polymorphism short tandem repeats could be detected for one or all three species as shown by sequencing analysis. In red deer, more than two alleles were found in eight microsatellites, in roe deer more than two alleles could be demonstrated in seven microsatellites, whereas in fallow deer more than two alleles were found in only two microsatellite polymorphism. A comparison of sequences of PCR products from the three deer species with the sequences of reindeer revealed several differences between the four species. In six microsatellites -- selected because or their reliability in PCR and because of their polymorphic character -- we established a sequenced allelic ladder and give population data of all three species from 82 deer of the Northeast region of Germany (Vorpommern). Our results show the possibility to use microsatellite polymorphism in the identification of deer in forensic applications like poaching.     

Identification of DNA of human origin based on amplification of human-specific mitochondrial cytochrome b region

Species-specific differences in a non-polymorphic region of the mitochondrial cytochrome b gene appear to be large enough to allow human-specific amplification of forensic DNA samples. We therefore developed a PCR-based method using newly designed primers to amplify a 157-bp portion of the human mitochondrial cytochrome b gene. The forward and reverse primers were designed to hybridize to regions of the human mitochondrial cytochrome b gene with sequences differing from those of chimpanzee by 26% (7 bp/27 bp) and 26% (6 bp/23 bp), respectively. Using this primer pair, we successfully amplified DNA extracted from blood samples of 48 healthy adults. All these human samples produced a single band of the expected size on agarose gel electrophoresis, and the sequence of the single band was shown to be identical to that of the target region (157 bp) by sequence analysis. On the other hand, no visible bands were amplified from DNA extracted from blood samples of animals including non-human primates (chimpanzee, gorilla, Japanese monkey, crab-eating monkey) and other species (cow, pig, dog, goat, rat, chicken and tuna). Thus, DNA producing a single band following PCR amplification using this primer pair can be reasonably interpreted as being of human origin. In addition, aged biological specimens comprising bloodstains, hair shafts and bones were successfully identified as being of human origin, illustrating the applicability of the present method to forensic specimens.     

用等电聚焦酶免疫分析法检测人类精斑GC表现型

本文用IEF结合使用过氧化物酶标记第二抗体的酶免疫分析法检测了17名键康成年男性的精液(斑)和唾液斑及10名健康成年女性的阴道分泌液中的GC表现型。结果发现17份人类精斑均可测出三种GC蛋白普通表现型。在10份阴道液中测出一份样本的GC表现型,3份样本有不甚清楚的GC带,不能定型,其他样本均无GC带。17份唾液斑未测出GC。本法的灵敏度(0.675ng)比文献报道的用过氧化物酶标记第二抗体的酶免疫分析(5.6ng)高。     

线粒体DNA cytb和HVI的分析用于种属鉴定

目的以线粒体DNA为目标序列,探讨生物检材的种属来源问题。方法复合扩增线粒体DNA细胞色素b基因(Cb)片段和D-环HVI上人源特异性DNA片段,2%琼脂糖凝胶电泳检测复合扩增产物谱带;用常规测序技术获得种属来源不明的检材Cytb基因序列,登陆美国国家生物信息中心网站主页(http://www.ncbi.nlm.nih.gov),将Cytb序列的测序结果用BLAST2.2.9[2004.5.1]进行匹配查询,查询数据库中存在的与其相匹配的物种条目。结果检材经复合扩增后电泳检测可区分人源性检材和非人源性检材;用生物信息法可确定检材种属来源结论检测线粒体DNA细胞色素b基因和D-环HVI的有关序列可在DNA分子水平上鉴别人源性检材和非人源性检材,结合测序的分子生物信息学方法,可对检材进行种属鉴定。     

Species identification in routine casework samples using the SPInDel kit

The identification of species in casework samples is of fundamental importance for forensic investigations. Laboratories are increasingly compelled to provide accurate and fast identifications in trace materials left on crime scenes, wildlife poaching, illegal trade of protected species, fraudulent food products cases, etc. However, the field of nonhuman forensic genetics is still working on the standardization of typing methods and practices. Here we describe the successful implementation of the Species Identification by Insertions/Deletions (SPInDel) method in routine casework analyses in 11 laboratories worldwide. The SPInDel was developed to detect human DNA, at the same time that identifies common animal species. The fragment size analysis of six mtDNA regions allows identification in suboptimal DNA samples, including mixtures, with no need for sequencing. The samples were collected from 2013 to 2018 and included hair, blood, meat, saliva, faeces, bones, etc. The SPInDel kit successfully identified >95% of the samples, being dog, human and pig the most frequently detected species. The six SPInDel loci were successfully amplified in mixtures and degraded samples (river water, sand, stains in clothes, etc.). Interestingly, several species that were not originally targeted by SPInDel primers were also identified (e.g., red fox, brown bear, fallow deer and red deer). In conclusion, the SPInDel kit was successfully used in crime scene investigations (often involving human DNA detection) and in cases of poaching, environmental contamination and food fraud. It is now becoming a useful tool for the routine analysis of nonhuman DNA samples within the high quality standards of forensic genetics.