利用荧光AFLP技术检测罂粟DNA多态性

目的 利用荧光AFLP检测技术检测罂粟植物DNA多态性。方法 用Axygen公司的AxyPrep DNA试剂盒提取了12株产于缅甸和中国云南省昆明市宜良县罂粟植株的DNA，用Eco RI和Mse I对总DNA进行酶切。连接人工接头，预扩增和选择性扩增，其产物在CEQ8000遗传分析系统上检测。结果 64对选择性扩增引物中8对引物能得到20条以上的扩增片断，具有高度多态性。结论 荧光AFLP技术可用于罂粟DNA多态性的检测。     

利用DNAITS2条形码序列鉴定植物大麻和罂粟

目的分析毒品原植物大麻、罂粟及其混伪品植物r DNA的ITS2序列信息,探索鉴定大麻、罂粟及其混伪品的新方法。方法采用PCR法扩增ITS2序列,双向测序后运用Codon Code Aligner、MEGA5.1软件进行数据处理,计算种内种间K2P距离,构建系统聚类树(NJ树)。结果大麻、罂粟的种内K2P遗传距离为0,大麻、罂粟及其混伪品之间的最小K2P距离为0.187。NJ树表现出明显的单系性。结论利用DNA ITS2条形码序列有可能鉴定出毒品原植物大麻、罂粟。     

利用种属特异性SSR荧光引物检出稀释液中罂粟DNA1例

正基于DNA检测技术建立的罂粟种属鉴别系统,能准确识别幼苗期罂粟、罂粟植株残渣、罂粟壳和罂粟种子,但针对涉毒案件中稀释液体所含罂粟的DNA种属检验目前尚无报道~([1-2])。本文应用种属特异性SSR荧光引物(simple sequence repeat,微卫星标记)的DNA检测技术,通过提取、扩增方法的优化,从1例涉毒案件送检的罂粟稀释浆液样本中检出SSR谱带并成功破案,为将来涉毒案件中类     

扩增12S rRNA基因鉴定生物检材种属

目的建立一种能够在同一反应条件下鉴定多个具体物种．又满足简单、快速、特异、灵敏、准确等实用要求的种属鉴定方法。方法从GenBank中获取人、鸡、鸭、鹅、猪、兔、鼠、绵羊、水牛、狗、山羊等11个物种的12SrRNA基因序列．设计一对针对上述11个物种的通用引物和分别针对人、鸡和鸭的特异引物,同时扩增各物种12SrRNA基因。以通用引物扩增片段为内部对照．以特异引物扩增片段用于人、鸡和鸭的种属鉴定,并分别对人、鸡、鸭单一检材以及人和鸡、人和鸭、鸡和鸭等二元混合DNA进行鉴定。结果通用引物扩增,各物种均有400bp左右的扩增片段：特异引物只对各自目标种属有扩增产物,片段大小：人163bp、鸡286bp、鸭374bp;测序结果与GenBank既有序列比对,Identities分值：人100％、鸡99％、鸭100％;通用引物扩增人、鸡和鸭检材的灵敏度为2．5Pg;特异引物扩增灵敏度人为2．5pg,鸡、鸭均为200Pg;混合DNA中任一种DNA的含量只要高于检测灵敏度即可被准确检出．不受另一种DNA量的干扰：盲测结果准确。结论本方法可以利用同一种PCR反应条件鉴定多个物种的种属。     

应用AFLP检测大麻遗传多样性

目的 筛选对于大麻多态性好的AFLP引物标记来区分大麻品种.方法 利用AFLP技术用55对引物组合对12个大麻地域品种进行初筛.结果 选出5对多态性好的引物组合进行了遗传多样性研究.每对AFLP引物组合扩增出47～76务带.共获得285条带,其中多态性条带为99条以及10条品种特异带.结论 AFLP对于大麻具有很高的分辨率,为今后深入地研究大麻植物遗传多样性奠定了很好的基础.     

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。     

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。     

IDentifier DNA分型盒（炎黄34）的法医学验证及应用评估

目的 测试IDentifier DNA分型盒(炎黄34)的技术性能指标,评估其法医学应用价值。方法 根据《法庭科学人类荧光标记STR复合扩增检测试剂质量基本要求》(GB/T 37226—2018),从种属特异性、分型准确性、灵敏度、适应性、耐受性、一致性、均衡性、反应条件验证、混合样本、稳定性、批间差11个方面对IDentifier DNA分型盒(炎黄34)进行测试。比较IDentifier DNA分型盒(炎黄34)与Power Plex^?Fusion 6C系统、Versa Plex^?27PY系统、Veri Filer^TM Plus PCR扩增试剂盒的系统效能。使用IDentifier DNA分型盒(炎黄34)检测日常案件中的拭子类生物检材,观察其STR检验结果 。结果 IDentifier DNA分型盒(炎黄34)具有良好的种属特异性、分型准确性、适应性、耐受性和均衡性,灵敏度可达0.062 5 ng,能检测案件中不同类型的检材、降解检材及混有抑制剂的检材,对混合比例为4∶1以内的样本均能获得完整分型。该试剂盒的系统效...     

线粒体16srRNA和ND4基因在种属鉴定中的应用研究

目的构建一种用于种属鉴定的线粒体DNA(m tDNA)16 srRNA和ND4基因荧光标记复合扩增检测体系。方法利用引物设计软件(Prim er 5)对两个m tDNA序列ND4基因和16 srRNA基因设计两对引物,每对引物中的一条在5’端标记荧光素(6-FAM)。按传统复合扩增技术建立复合扩增体系,用AB I PR ISM 310基因分析仪对产物进行分析。结果人类DNA扩增产物出现两个峰,片段大小分别为110bp的人类特异片段和149bp的人与动物共有片段,而动物DNA扩增产物出现一个峰,片段大小为149bp。对30个实验室存放5~15年的陈旧人血痕也能明确判断其种属来源。结论该体系可以明确区分人源性生物检材与其它常见动物样本,对实验室长期存放的陈旧检材也具有较好的检测能力。     

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。     

TD-RAPD技术用于罂粟品种鉴定初探

目的探索TD-RAPD技术用于罂粟品种鉴定的可行性。方法采用改良CTAB法从罂粟叶片中提取DNA;TD-RAPD技术对种植于云南西双版纳地区的1个罂粟样品进行扩增分析。结果建立了罂粟DNA提取方法,从10个随机引物中筛选出6个引物用于罂粟TD-RAPD分析。结论TD-RAPD技术可用于罂粟DNA的分子标记,为罂粟DNA数据库建立提供技术方法,最终从DNA分子水平上追溯罂粟植物毒源。     

DNA polymorphism detection of Papaver somniferum L using fluorescent amplified fragment length polymorphism

OBJECTIVE: To detect DNA polymorphism of Papaver somniferum L using fluorescent Amplified Fragment Length Polymorphism. METHODS: Genomic DNA was isolated using the AxyPrep DNA Kit, double-digested by two restrictional endonucleases (Eco RI and Mse I) and ligated to oligonucleotide adapters. After Pre-amplification and selective amplification, the DNA fragments were separated by capillary electrophoresis using the CEQ8000 DNA Fragment Analyzer. RESULTS: More than 20 fragments of highly polymorphic products were obtained by 8 pairs of primer from 64 selective amplifying primer pairs. CONCLUSION: The fluorescent AFLP technique can be used to detect the DNA polymorphism of Papaver somniferum.     

Discrimination among species of Papaver based on the plastid rpl16 gene and the rpl16-rpl14 spacer sequence

An attempt was made to discriminate among six species of Papaver (P. bracteatum, P. orientale, P. pseudo-orientale, P. rhoeas, P. setigerum and P. somniferum) by comparing the nucleotide sequences of the plastid rpl16 gene and the rpl16-rpl14 spacer region. Comparison of sequences allowed us to distinguish five species, namely P. bracteatum, P. orientale, P. pseudo-orientale, P. rhoeas and P. setigerum plus P. somniferum from one another, but sequences from P. setigerum and P. somniferum were identical. It is difficult to distinguish between P. bracteatum, P. orientale and P. pseudo-orientale at the vegetative stage of growth. However, our method allowed us to distinguish between these three species and the others using nucleotide sequences and should allow identification of P. bracteatum that has been cultivated illegally in the garden in Japan. Furthermore, P. rhoeas was clearly discriminated from P. setigerum and P. somniferum by reference to the sequence of the rpl16 exon using young seedlings.     

毒品原植物 罂粟与其近缘物种的形态学差异解析

目的构建能快速准确鉴别毒品原植物罂粟的形态学指标。方法本研究收集考察了不同来源的毒品原植物罂粟与其近缘物种,并进行同园栽培实验,通过对毒品原植物罂粟及其近缘物种形态特征的观察与测量,分析可以用于精准、快速鉴别毒品原植物罂粟与其近缘物种的稳定形态特征。结果筛选出叶形态作为鉴别幼苗期毒品原植物罂粟的形态特征;株高、毛被、叶形态和蒴果形态作为鉴别花果期毒品原植物罂粟的形态特征。结论形态学是植物分类鉴定的重要依据,该套形态学指标将有力推动毒品原植物罂粟的快速鉴别,有效提升毒品原植物罂粟种植案件的侦破能力。     

Exploiting expressed sequence tag databases for the development and characterization of gene-derived simple sequence repeat markers in the opium poppy (Papaver somniferum L.) for forensic applications

Simple sequence repeat (SSR) markers in the opium poppy (Papaver somniferum L.) were identified from an expressed sequence tag (EST) database comprised of 20,340 sequences. In total, 2780 SSR-containing sequences were identified. The most frequent microsatellite had an AT/TA motif (37%). Twenty-two opium poppy EST-SSR markers were presently developed and polymorphisms of six markers (psom 2, 4, 12, 13, 17, and 22) were utilized in 135 individuals under narcotic control investigation. An average of three alleles per locus (range: 2-5 alleles) with a mean heterozygosity of 0.167 was detected. Six loci identified 29 unique profiles in 135 individuals. The EST-SSR markers exhibited small degrees of genetic differentiation (fixation index = 0.727, p < 0.001). Other variable markers will be needed to facilitate the forensic identification of the opium poppy for future cases. To determine the potential for cross-species amplification, six markers were tested in five Papaver genera species and two Eschscholzia genera. The psom 4 and psom 17 primer pair was transferable. This is the first study to report SSR markers of the opium poppy.     

Genetic and chemical components analysis of Papaver setigerum naturalized in Korea

Of the 110 species of genus Papaver, only Papaver somniferum and P. setigerum are controlled poppies in Korea. All poppy samples share similar morphology therefore it is important to check if they contain controlled substances such as morphine and codeine for forensic purpose. Since the alkaloid content of Papaver plants varies according to their growing stage, chemical components analysis alone is not enough to identify exact species. In 2010, hundreds of poppy plants suspected to be P. somniferum were found in Jeju Island, South Korea. They had a slightly different but overall similar appearance to P. somniferum. Using GC-MS analysis, codeine, rhoeadine, papaverine, protopine, noscapine, setigeridine and trace amounts of morphine were detected in these samples. Although their chemical components were different from what has been described in literatures for P. setigerum, they could be assumed to be P. setigerum based on their morphological features and GC-MS results. Also, chromosome numbers using their seeds showed 2n=44 and the numbers were in accordance with those of P. setigerum. Nucleotide substitution or insertion/deletion of ITS (internal transcribed spacer), 18S rRNA (ribosomal RNA), rbcL (large subunit of ribulose 1,5-bisphosphate carboxylase), trnL-trnF IGS (intergenic spacer), trnL intron and psbA-trnH were assessed as universal genetic markers for P. setigerum. Also, genetic analysis using six target genes involved in the biosynthesis of benzylisoquinoline alkaloids, including TYDC (tyrosine/dopa decarboxylase), SAT (salutaridinol-7-O-acetyltransferase), BBE (berberine bridge enzyme), COR (codeinone reductase), CYP80B1 ((S)-N-methylcoclaurine 3'-hydroxylase) and NCS (norcoclaurine synthase) were tested as Papaver-specific genetic markers by the existence of their PCR products. From the results, the sequences of the 6 universal genetic markers and 6 Papaver-specific genetic markers for P. setigerum were identified and then Genbank accession numbers of them were registered in NCBI. Also, the trnL intron and psbA-trnH nucleic acid sequences of the 7 Papaver species were identified and registered.     

Identification of Cannabis sativa L. (Cannabaceae) using restriction profiles of the Internal Transcribed Spacer II (ITS2)

The paper describes the construction of a restriction profile of the Internal Transcribed Spacer II (ITS2) of Cannabis sativa L. Nuclear Ribosomal DNA, using digestion by a selected number of restriction endonucleases. The method was evaluated for false positives and it is suggested that it could be used to identify suspected Cannabis samples, by comparing their restriction profiles with the restriction profile of known Cannabis material.     

Preliminary data on the usefulness of internal transcribed spacer I (ITS1) sequence in Cannabis sativa L. identification.

A method is described to identify an unknown sample of plant material of forensic interest as Cannabis sativa L. The method consists in comparing the sequence of the nuclear ribosomal DNA Internal Transcribed Spacer I (ITS1) of the unknown sample with a Cannabis sequence. Our preliminary results show that the ITS1 is an ideal molecule for the identification of a sample suspected to be marijuana.     

Genetic variation in hemp and marijuana (Cannabis sativa L.) according to amplified fragment length polymorphisms

Cannabis sativa L. (Cannabaceae) is one of the earliest known cultivated plants and is important in the global economy today as a licit and an illicit crop. Molecular markers distinguishing licit and illicit cultivars have forensic utility, but no direct comparison of hemp and marijuana amplified fragment length polymorphism (AFLP) has been made to date. Genetic variation was surveyed in three populations of fiber hemp and a potent cultivar of marijuana using AFLP markers. Ten primer pairs yielded 1206 bands, of which 88% were polymorphic. Eighteen bands represented fixed differences between all fiber populations and the drug cultivar. These markers have practical utility for (1) establishing conspiracy in the cultivation and distribution of marijuana, (2) identifying geographic sources of seized drugs, and (3) discriminating illegal, potent marijuana cultivars from hemp where the cultivation of industrial hemp is permitted.