基于28SrRNA基因序列对洛阳地区嗜尸性蝇类的分子鉴定CSCD

目的通过检测嗜尸性蝇类28S r RNA基因中715 bp序列,鉴定常见嗜尸性蝇类种属,解决其形态学鉴定难题,为死亡时间推断提供技术支持。方法收集洛阳地区常见嗜尸性蝇类标本29只,经形态学鉴定后,用Chelex-100法提取腿部DNA,并对28S r RNA基因片段进行扩增和测序,与Gen Bank和EMBL数据库中的28条相应蝇种序列进行比对,用MEGA7.0软件进行序列整理,通过BLAST搜索进行序列比对,并分析所得序列碱基组成,建立种内及种间进化分歧率,构建系统发育树。结果形态学鉴定29只嗜尸性蝇类归属于3科5属6种。获得28S r RNA基因中715 bp的序列,在线BLAST比对结果显示相似度100%。系统发育树显示5种蝇类可以较好聚类。不同蝇种种间差异0.007~0.045,种内差异0~0.001,种间差异和种内差异没有交叉。结论 28S r RNA靶基因序列片段对嗜尸性蝇类有良好的鉴别能力,可以作为新的嗜尸性蝇类种属鉴定遗传标记。     

基于28SrRNA基因序列对洛阳地区嗜尸性蝇类的分子鉴定

目的通过检测嗜尸性蝇类28S r RNA基因中715 bp序列,鉴定常见嗜尸性蝇类种属,解决其形态学鉴定难题,为死亡时间推断提供技术支持。方法收集洛阳地区常见嗜尸性蝇类标本29只,经形态学鉴定后,用Chelex-100法提取腿部DNA,并对28S r RNA基因片段进行扩增和测序,与Gen Bank和EMBL数据库中的28条相应蝇种序列进行比对,用MEGA7.0软件进行序列整理,通过BLAST搜索进行序列比对,并分析所得序列碱基组成,建立种内及种间进化分歧率,构建系统发育树。结果形态学鉴定29只嗜尸性蝇类归属于3科5属6种。获得28S r RNA基因中715 bp的序列,在线BLAST比对结果显示相似度100%。系统发育树显示5种蝇类可以较好聚类。不同蝇种种间差异0.007~0.045,种内差异0~0.001,种间差异和种内差异没有交叉。结论 28S r RNA靶基因序列片段对嗜尸性蝇类有良好的鉴别能力,可以作为新的嗜尸性蝇类种属鉴定遗传标记。     

利用16S rDNA序列鉴定常见嗜尸性蝇类种属

目的通过分析16S rDNA 551bp基因序列,鉴定常见嗜尸性蝇类种属。方法随机采集17个地区放置于室外草地的家兔尸体上7个种24只嗜尸性苍蝇样本,经形态学鉴定种类后,提取胸肌DNA,对16S rDNA 551bp基因片段进行PCR扩增,产物纯化、测序后上传GenBank;利用MEGA 4.0软件构建序列间的系统发育树,分析建立种内及种间进化分歧表。结果 24只样本16S rDNA序列分析显示7种蝇类可以较好聚类;其中棕尾别麻蝇种内进化分歧整体均数为2.8%,家蝇为1.5%,丽蝇科的5个种均在0.7%以内。上述7个蝇种的种间进化分歧均数在1.6%～7.1%之间。其中,棕尾别麻蝇、家蝇与其它蝇类的种间分歧均数在4.0%～7.1%之间。结论本文分析结果显示,蝇种间同源性相差明显,采用mtDNA 16S rDNA中551bp基因序列分析,可进行蝇种鉴定。     

mtDNA COI和ND5基因用于鉴别常见嗜尸性蝇类

目的对蝇类mtDNA 523bp COI和347bp ND5基因片段进行序列分析,评价其在以嗜尸性蝇类种属鉴定中应用的可行性。方法从广州(广东省)、湛江(广东省)、韶关(广东省)、沈丘(河南省)及蜂蛹寨(四川省)采集7种嗜尸性蝇类标本,进行形态学种属鉴定,取其腹部肌肉提取DNA,利用基因特异性引物对线粒体COI、ND5基因进行PCR扩增,产物经纯化后进行测序,MEGA 3.0软件对DNA序列进行碱基组成、进化分歧率和系统发育分析。结果进化分歧率ND5基因种内小于1.83%,种间大于2.62%;种间与种内进化分歧率范围间没有交叉;COI基因种间在0.48%～14.8%之间,种内在0.24%～8.3%之间,种内进化分歧范围与种间进化分歧范围存在交叉。结论 ND5基因片段可在种水平有效鉴别常见嗜尸性蝇类,也可鉴别近缘种。而单独运用COI基因不能有效进行种属鉴定。     

高分辨率熔解曲线检测常见嗜尸性丽蝇

目的 建立高分辨率熔解曲线（HRM）鉴定常见嗜尸性丽蝇的检测方法,以期为嗜尸性蝇类的分子鉴定提供依据和技术支持。方法 收集洛阳地区常见嗜尸性丽蝇标本26只,经形态学鉴定后,提取腿部DNA,基于COI基因进行PCR-HRM检测,对所得HRM图谱和解链温度（Tm）数据进行分析和统计,DNA测序以验证HRM检测结果。结果 根据熔解曲线形状和解链温度可将本研究中的26只嗜尸性丽蝇归属于5属8种,与形态学鉴定、DNA测序结果完全一致。结论 基于COI片段的HRM技术是一种便捷有效的检测方法,可用于嗜尸性蝇类的快速鉴定。     

提取CO Ⅰ基因片段鉴定嗜尸性蝇类

目的通过扩增蝇类COⅠ基因片段,结合形态学特征鉴定嗜尸性蝇类的种类。方法运用改良平衡酚—tris饱和酚法提取蝇类DNA,进行COⅠ序列扩增和测序,与数据库序列进行比对和分析。结果改良平衡酚—tris饱和酚提取DNA方法可获得有效的蝇类DNA,并可应用于COⅠ基因片段扩增,进而进行蝇类种类的鉴定。结论平衡酚—Tris饱和酚法提取的噬尸性蝇类虫体DNA作为模板,用于COⅠ序列扩增,测序后与数据库目的序列分析比对,可准确鉴定嗜尸性蝇类的种类;和传统的昆虫形态学特征鉴定方法比较,该体系更准确,应用范围更广。     

应用mtDNA COI基因序列鉴别常见嗜尸性蝇类种属

目的探讨mtDNA基因序列对常见嗜尸性蝇类的种属鉴别应用价值。方法收集不同区域2科4属6个种30个蝇类样本,提取样本线粒体DNA后扩增COI基因序列,以琼脂糖电泳检测扩增产物并测序,以DNAMAN6.0分析软件分别截取498bp序列,用MEGA5.2软件分别进行序列分析,然后构建系统发育树,比较各地区不同种属样本的序列差异。结果 6个种属的嗜尸性蝇类30个样本mtDNA的COI基因具有一定的序列差异,种内进化分歧均数在0.1%~1.6%之间,种间进化分歧均数在2.2%~11.2%之间,6个种属通过系统发育树均可明确区分。结论 COI基因序列分析和系统发育树对嗜尸性蝇类的种属检验具有重要帮助作用,可用于现场样本的准确、快速种属鉴定。     

贵阳地区常见尸食性蝇类mtDNA分子标记的检测

目的应用mt DNA中COI(348bp)、COII(637bp)及16Sr DNA(513bp)基因序列鉴定贵阳地区常见尸食性蝇类的种属。方法收集贵阳地区常见尸食性蝇类标本,经形态学分类鉴定后提取胸肌DNA,扩增COI(348bp)、COII(637bp)及16Sr DNA(513bp)基因片段,测序后用DNAMAN 4.0序列分析软件进行序列拼接,NCBI中的BLAST、MEGA 5.2软件包对所得序列分析建立种内及种间进化分歧率,并构建系统发育树。结果 COI种间平均进化分歧率为12.55%,种间范围在3.0%~18.6%,种内范围在0%~0.7%;COII种间平均进化分歧率为13.73%,种间范围在5.3%~21.6%,种内范围在0%~0.6%;16Sr DNA种间平均进化分歧率为4.54%,种间范围在0.4%~7.6%,种内范围在0%~0.4%。结论 COI、COII及16Sr DNA可用于尸食性蝇类的种属鉴定,该方法可作为传统分类方法的补充手段。     

利用COI序列序列鉴定常见嗜尸性蝇种

目的 检测嗜尸性蝇类线粒体DNA(mtDNA)细胞色素氧化酶辅酶Ⅰ(COI)中278bp序列,鉴定蝇科3属5种嗜尸性蝇,解决其形态学种类鉴定的难题,为死亡时间推断提供技术支持.方法 从15省、市(地区)室外草地家兔尸体上采集蝇科3属5种共计18个蝇类成蝇样本,提取mtDNA进行PCR扩增,序列测定且上传GENBANK,...     

郑州地区食尸性苍蝇及幼虫mtDNA中COⅡ基因序列分析

目的 用mtDNA中细胞色素氧化酶辅酶Ⅱ(COⅡ)基因序列鉴定法医学中常见食尸性苍蝇及其幼虫的种类. 方法 收集郑州地区大白鼠尸体上的苍蝇及其幼虫,提取DNA,PCR扩增CO Ⅱ序列,测序,用Clustalx和MEGA 4.0软件对基因序列进行比对分析及构建系统进化树.结果 成虫与幼虫基因差异不明显,CO Ⅱ基因序列可以对棕尾别麻蝇、巨尾阿丽蝇和亮绿蝇进行鉴定,铜绿蝇与丝光绿蝇进化距离较近,CO Ⅱ序列不能将他们区分开,同时还发现巨尾阿丽蝇和亮绿蝇存在种群单核苷酸多态性. 结论 mtDNA中COⅡ序列能有效鉴定郑州地区部分常见食尸性苍蝇的种类,方法 简便、准确.     

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。     

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.     

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.     

Identification of mammal species using species-specific DNA pyrosequencing

In forensic casework it is highly relevant to be able to deduce the species origin of an unknown biological sample. For such a purpose we have designed and developed an assay for species identification based on DNA sequencing of two short mitochondrial DNA amplicons. In short, partial 12S rRNA and partial 16S rRNA fragments (approximately 100bp) are amplified by PCR followed by direct sequencing using pyrosequencing technique. Due to properties of the chosen targets, the same PCR conditions and primers were used irrespective of the true species of an unknown sample. A total of 28 different mammals present in the European fauna were sequenced both for the partial 12S rRNA and the partial 16S rRNA sequences for accuracy verification. Together the two sequences showed to have a high divergence factor, discriminating almost all mammals. Furthermore, the human reference nucleotide sequences were always at least nine nucleotides different compared to the other sequenced species both at the partial 12S rRNA and the partial 16S rRNA sequences.     

16SrDNA序列分析在嗜尸性蝇类鉴定中的应用

目的通过mtDNA上16SrDNA中551bp基因序列分析,解决依据COI和COII上278bp和635bp基因序列难以鉴别丝光绿蝇、铜绿蝇种类的难题,为法医学嗜尸性苍蝇种类的鉴别提供依据。方法随机采集放置在呼和浩特及成都地区室外草地家兔尸体上的铜绿蝇和丝光绿蝇,对其mtDNA上16SrDNA中551bp基因序列进行分析、比对,构建系统发育树。结果通过对嗜尸性蝇类mtDNA上16SrDNA的551bp基因序列分析可以对丝光绿蝇和铜绿蝇进行鉴别。结论16SrDNA上551bp基因序列分析是对嗜尸性蝇类(尤其是铜绿蝇和丝光绿蝇)进行种类鉴定的有效方法,是对依据COⅠ和COⅡ序列分析方法鉴别嗜尸性苍蝇种类的重要补充。     

潍坊地区六种常见嗜尸性蝇类mtDNA COI区序列研究

目的解决蝇类成虫、蛹和蛆快速、准确鉴定的难题。方法随机采集潍坊地区人尸周围环境中的蝇类成虫、蛹和蛆,提取其mtDNA,经PCR扩增、产物检测与纯化、测序等,比较其序列差异。结果六种常见嗜尸性蝇类相互之间序列差异明显,同一种蝇的成虫、幼虫、蛹之间未见差异。结论mtDNACOI区序列分析可作为法医学嗜尸性蝇类种属鉴别的可靠方法。     

Molecular Identification of Necrophagous Dermestes Species in South Korea Using Cytochrome c Oxidase Subunit I Nucleotide Sequences (Genus Dermestes)

Species identification of necrophagous insects found on a dead body is an essential key in applying medicolegal entomology to the estimation of postmortem interval (PMI). Due to limited morphological identification of insect evidence, several studies have identified species using molecular information such as DNA markers. While considerable cytochrome c oxidase subunit I (COI) gene sequence data of necrophagous fly species have been collected and annotated, those of necrophagous beetle species have not. Since necrophagous beetles such as Dermestes species have a larval period longer than that of flies, beetles are useful in even the late decomposition phase in estimating minimum PMI. To obtain the full-length COI gene sequences of six Dermestes species collected from South Korea, we designed primers for polymerase chain reaction amplification and sequencing. The obtained full COI nucleotide sequences were used for performing phylogenic analysis and comparison with previously reported sequences. The results demonstrated that the COI gene sequences could be used to identify forensically important Dermestes species in South Korea.