COⅠ基因微条形码技术在毛发种属鉴定中的应用

目的利用COⅠ基因微条形码技术对哺乳动物毛发进行种属鉴定。方法设计一对哺乳动物COⅠ基因微条形码通用引物,利用共同区PCR技术对来自于哺乳纲5个目11个种属的实验动物毛发DNA进行扩增,并对扩增产物进行双向引物测序,将测序结果进行拼接后所得的基因序列输入BOLD数据库进行同源性比对。结果本研究设计的微条形码通用引物能够对所有种属实验动物毛发DNA进行扩增,扩增片段长度147 bp。数据库同源比对结果显示最匹配物种与实验动物种属相符,除狮同源匹配度为98.99%外,其他实验动物同源匹配度均为100%,且狮种内遗传距离1%,种间遗传距离大于种内遗传距离十倍,可以进行种属判定。结论建立的COⅠ基因微条形码技术能够快速准确地对哺乳动物毛发检材进行种属鉴定。     

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。     

DNA分析与种属鉴定

种属鉴定在法医学实践中占有重要地位。现行用于法医学种属鉴定的方法主要有形态学、血清学和DNA分析技术。本文就种属鉴定的技术发展以及各种技术的优缺点做一比较,重点讨论了DNA分析技术在种属鉴定中的应用价值。     

植物DNA条形码技术用于交通命案的调查

目的以rDNA-ITS序列片段为植物DNA条形码,对涉及一例交通命案的植物检材进行种属鉴定,判断三轮车左侧车体上提取的树皮样物质与农用车所运的树木是否为同一树种。方法采用改良CTAB法提取植物DNA,先将DNA纯化,再进行rDNA-ITS的PCR扩增,然后对PCR产物切胶、回收,克隆、测序,对测序结果进行BLAST分析,并构建系统发育树。结果三轮车上粘附的树皮来源于柑橘（Citrus sunki）,农用车所运的树木为界山三角槭（Acer buergerianum）,两者非同一种属。结论用rDNA-ITS序列片段用作植物DNA条形码对植物种属进行鉴定,可获得可靠的鉴定意见。     

采用DNA条形码技术鉴定常见杨树种属初探

目的应用DNA条形码技术鉴定杨树种属,为建立现场植物物证种属鉴定方法提供参考信息。方法收集15种常见杨树叶样本,利用DNA条形码技术对ITS2、mat K和rbc L 3条序列进行测序和物种鉴定效率分析,使用MEGA 5软件进行K2P遗传距离和聚类分析。结果除mat K外,其余两条候选片段的PCR扩增及测序效率均为100%;鉴定效率ITS2为53%、mat K为50%、rbc L3为17.9%;以序列组合ITS2+mat K对15种常见杨树在物种水平的鉴定效率为100%。15种杨树ITS2序列中巨霸杨与山杨种间K2P遗传距离最大,为0.047 9;mat K序列中河北杨、小黑杨、新疆杨与银白杨的种间K2P遗传距离最大,均为0.009 0;结论 ITS2+mat K序列组合可以鉴定15种不同种杨树种类,并有望作为杨树品种鉴定的潜在条形码组合。     

利用DNA条形码技术鉴定木材种属1例

正目前,国内刑事技术领域涉及木材DNA种属鉴定的方法尚属空白。在盗砍盗伐、非法走私、假冒仿造木材类案件中,常因木材种类无法判定造成案件无法侦办和诉讼。白木香(Aquilaria sinensis)又称沉香,为桃金娘目(Myrtales)瑞香科(Thymelaeaceae)沉香属(Aquilaria),是木材市场炙手可热的木材种类。近几年,DNA条形码(DNA barcoding)[1-2]技术发展迅速,     

基于ITS2条形码序列对新型“香料”毒品植物成分的研究

目的应用DNA条形码片段ITS2对新型毒品样品中的植物成分进行分析。方法提取可疑毒品样本"spike99","K2","7号"中的植物基因组DNA,用ITS2通用引物进行扩增,对扩增产物进行测序,对序列校对拼接,应用BLAST方法在NCBI数据库中比对。结果 "spike 99"、"7号"各得到1条ITS2基因序列,"K2"得到2条ITS2基因序列,分别与紫花苜蓿、大麻、啤酒花、黄蜀葵的ITS2基因序列的同源性为100%。结论应用条形码技术可以成功分析新型毒品中的植物种属,为案件中植物样本的种属鉴定提供方法。     

利用DNA条形码技术鉴别植物

目的研究利用DNA条形码技术鉴定上海地区常见植物叶片种类,在物种鉴定水平的基础上,进一步讨论利用分子标记技术对该类物证进行同一认定可行性。方法随机选取上海地区常见的9种植物,在其非新鲜的状态下利用高盐法提取植物组织的DNA,PCR扩增叶绿体rbcL和matK基因片段并且回收测序,通过数据库比对确定植物的物种。结果利用双基因鉴定分析系统,在随机收集的9份样品中,均达到物种水平的鉴定。结论利用DNA条形码技术,可以在物种水平准确地鉴定植物叶片类物证。     

DNA条形码技术在法医昆虫学中的研究进展

嗜尸性昆虫的准确鉴定是应用昆虫判断死亡时间的先决条件,但是昆虫的鉴定只有相关类群的昆虫专家能够鉴定。DNA条形码（DNA Barcoding）技术是利用一个或少数几个DNA片段对地球上现有物种进行识别和鉴定的一项新技术。这一技术给生物分类研究带来了空前的繁荣,同时也给法医昆虫学中各昆虫种类的鉴别研究带来新的动力。     

PCR扩增SON基因3’非编码区进行种属鉴定

根据人 DNA的 SON基因碱基序列选择性设计一对特异性引物 ,并对人和猕猴、阿拉伯狒狒、猪、牛、羊、马、驴、骡、狗、猫、兔、大白鼠、小白鼠、豚鼠等 14种哺乳类动物染色体 DNA的 SON基因 3’非编码区中的种属特异性区域进行 PCR扩增 ,扩增产物经 SSCP分离银染色技术检测 ,观察到人和 14种哺乳类动物的 SSCP图谱有明显不同。本方法可以对人和上述 14种哺乳类动物进行种属鉴定 ,适合于法医学种属鉴定的应用     

Identification of Indian Crocodile Species Through DNA Barcodes

The biodiversity of India includes three crocodile species, Crocodylus palustris, Crocodylus porosus, and Gavialis gangeticus, whose status is threatened due to bushmeat crisis and illegal hunting. The crocodilian conservation management requires novel techniques to help forensic analysts to reveal species identity. DNA barcoding is a species identification technique, where a partial cytochrome c oxidase subunit 1 gene is used as a marker for species identification. Herein, the DNA barcoding technique is evaluated for three Indian crocodiles by analyzing an approximately 750‐bp barcode region. The alignment result shows interspecific variations between sequences for discrimination of the three Indian crocodiles leading to species identification. The phylogenetic analyses also substantiate the established crocodilian relationships, which add further advantage to use this DNA barcoding approach for Indian crocodiles. This study provides preliminary evidences for the use of DNA barcoding technique in the identification of Indian crocodile species.     

DNA Barcoding in Forensic Entomology – Establishing a DNA Reference Library of Potentially Forensic Relevant Arthropod Species,

Throughout the years, DNA barcoding has gained in importance in forensic entomology as it leads to fast and reliable species determination. High‐quality results, however, can only be achieved with a comprehensive DNA barcode reference database at hand. In collaboration with the Bavarian State Criminal Police Office, we have initiated at the Bavarian State Collection of Zoology the establishment of a reference library containing arthropods of potential forensic relevance to be used for DNA barcoding applications. CO1‐5P’ DNA barcode sequences of hundreds of arthropods were obtained via DNA extraction, PCR and Sanger Sequencing, leading to the establishment of a database containing 502 high‐quality sequences which provide coverage for 88 arthropod species. Furthermore, we demonstrate an application example of this library using it as a backbone to a high throughput sequencing analysis of arthropod bulk samples collected from human corpses, which enabled the identification of 31 different arthropod Barcode Index Numbers.     

洛阳地区5种嗜尸性麻蝇分子鉴定研究

目的应用经典DNA条形码和28S r RNA基因鉴定洛阳地区5种常见嗜尸性麻蝇种属,评价其在常见嗜尸性麻蝇种属鉴定中应用的可行性。方法收集洛阳地区常见嗜尸性麻蝇类标本18只,经形态学分类鉴定后,Chelex-100法提取足部DNA,扩增并测序线粒体细胞色素C氧化酶亚基I(COI)和28S核糖体核糖核酸(28S r RNA)基因片段,BLAST比对后,与来自DNA条形码数据库(Barcode of Life Data,BOLD)的中国和韩国地区20条相应蝇种序列进行比对,用MEGA 7.0软件整理分析所得序列,计算碱基组成、种内及种间进化分歧率,并构建系统发育树。结果形态学鉴定18只嗜尸性麻蝇归于3属5种。每个样本获得COI和28S r RNA的扩增长度分别为646bp和721bp。在线BLAST比对结果显示样本COI序列相似度99%以上,邻近法构建发育树Bootstrap值为1 000,5种麻蝇可以较好聚类,与形态学鉴定结果一致。38个样本COI种内范围为0~0.022,棕尾别麻蝇、酱亚麻蝇和急钩(亚)麻蝇种间范围在0.057~0.090;赤尾麻蝇和红尾粪麻蝇种间范围在0~0.086。5种麻蝇的28S r RNA序列发育树显示,棕尾别麻蝇和急钩(亚)麻蝇明显各自聚类,其余3种成一类。结论对于本研究中的5种嗜尸性麻蝇,基于COI基因的DNA条形码可以有效区分棕尾别麻蝇、酱亚麻蝇和急钩(亚)麻蝇,28S r RNA基因只可以区分棕尾别麻蝇,二者可作为形态学鉴定之外的重要方法补充。     

A Three‐Locus,PCR‐based Method for Forensic Identification of Plant Material

Plant residue is currently an underutilized resource in forensic investigations despite the fact that many crime scenes, as well as suspects and victims, harbor plant‐derived residue that could be recovered and analyzed. Notwithstanding the considerable skill of forensic botanists, current methods of species determination could benefit from tools for DNA‐based species identification. However, DNA barcoding in plants has been hampered by sequence complications in the plant genome. Following a database search for usable barcodes, broad‐spectrum primers were designed and utilized to amplify and sequence the rbcL, trnL‐F, and rrn18 genetic loci from a variety of household plants. Once obtained, these DNA sequences were used to design species‐targeted primers that could successfully discriminate the source of plant residue from among the 21 species tested.     

Validation of the barcoding gene COI for use in forensic genetic species identification

The application of forensics to wildlife crime investigation routinely involves genetic species identification based on DNA sequence similarity. This work can be hindered by a lack of authenticated reference DNA sequence data resulting in weak matches between evidence and reference samples. The introduction of DNA barcoding has highlighted the expanding use of the mtDNA gene, cytochrome c oxidase I (COI), as a genetic marker for species identification. Here, we assess the COI gene for use in forensic analysis following published human validation guidelines. Validation experiments investigated reproducibility, heteroplasmy, mixed DNA, DNA template concentration, chemical treatments, substrate variation, environmental conditions and thermocycling parameters. Sequence similarity searches using both GenBank BLASTn and BOLD search engines indicated that the COI gene consistently identifies species where authenticated reference sequence data exists. Where misidentification occurred the cause was attributable to either erroneous reference sequences from published data, or lack of primer specificity. Although amplification failure was observed under certain sample treatments, there was no evidence of environmentally induced sequence mutation in those sequences that were generated. A simulated case study compared the performance of COI and cytochrome b mtDNA genes. Findings are discussed in relation to the utility of the COI gene in forensic species identification.     

Forensic Identification of CITES Protected Slimming Cactus (Hoodia) Using DNA Barcoding

Slimming cactus (Hoodia), found only in southwestern Africa, is a well‐known herbal product for losing weight. Consequently, Hoodia extracts are sought‐after worldwide despite a CITES Appendix II status. The failure to eradicate illegal trade is due to problems with detecting and identifying Hoodia using morphological and chemical characters. Our aim was to evaluate the potential of molecular identification of Hoodia based on DNA barcoding. Screening of nrITS1 and psbA‐trnH DNA sequences from 26 accessions of Ceropegieae resulted in successful identification, while conventional chemical profiling using DLI‐MS led to inaccurate detection and identification of Hoodia. The presence of Hoodia in herbal products was also successfully established using DNA sequences. A validation procedure of our DNA barcoding protocol demonstrated its robustness to changes in PCR conditions. We conclude that DNA barcoding is an effective tool for Hoodia detection and identification which can contribute to preventing illegal trade.     

Forensic Identification of Indian Snakeroot (Rauvolfia serpentina Benth. ex Kurz) Using DNA Barcoding

Indian snakeroot (Rauvolfia serpentina) is a valuable forest product, root extracts of which are used as an antihypertensive drug. Increasing demand led to overharvesting in the wild. Control of international trade is hampered by the inability to identify root samples to the species level. We therefore evaluated the potential of molecular identification by searching for species‐specific DNA polymorphisms. We found two species‐specific indels in the rps16 intron region for R. serpentina. Our DNA barcoding method was tested for its specificity, reproducibility, sensitivity and stability. We included samples of various tissues and ages, which had been treated differently for preservation. DNA extractions were tested in a range of amplification settings and dilutions. Species‐specific rps16 intron sequences were obtained from 79 herbarium accessions and one confiscated root, encompassing 39 different species. Our results demonstrate that molecular analysis provides new perspectives for forensic identification of Indian snakeroot.     

Molecular identification of forensically important fly species in Spain using COI barcodes

Species identification with DNA barcodes has been proven to be effective on different organisms and, particularly, has become a routinely used and quite accurate tool in forensic entomology to study necrophagous Diptera species. In this study, we analysed 215 specimens belonging to 42 species of 17 genera, from 9 different Diptera families. Flies were collected in 39 Spanish localities of the Iberian Peninsula sampled across three years in the four seasons. Intraspecific variation ranged from 0 to 2.46% whereas interspecific variation fluctuated from 3.07 to 14.59%, measuring 651 pb of the cytochrome oxidase subunit one (COI) gene. Neighbour-Joining analysis was carried out to investigate the molecular identification capabilities of the barcoding region, recovering almost all species as distinct monophyletic groups. The species groupings were generally consistent with morphological and molecular identifications. This work, which is the first with this intensive and extensive sampling in this area, shows that the COI barcode is an appropriate marker for unambiguous identification of forensically important Diptera in Spain.