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。     

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.     

嗜尸性苍蝇mtDNA中COⅡ基因序列检测

目的利用线粒体DNA(m tDNA)上细胞色素氧化酶辅酶Ⅱ(COⅡ)中635bp基因序列,解决嗜尸性苍蝇及其卵和幼虫种类鉴定的难题。方法随机采集放置在呼和浩特地区室外草地家兔尸体上的嗜尸性苍蝇、幼虫、苍蝇腹中的卵。利用Chelex方法提取上述苍蝇m tDNA;通过Perk in-E lm er 9600扩增仪进行PCR扩增;琼脂糖水平电泳和银染显色技术进行扩增结果检测;PCR胶回收试剂盒纯化;AB I 377测序仪测序;DNAMAN 4.0序列分析软件,进行序列比对,截取等长度片段;MEGA2.1软件包进行序列分析和构建系统发育树。结果上述嗜尸性苍蝇m tDNA上COⅡ基因序列在双翅目嗜尸性苍蝇的种内差异均数小于1%,种间差异均数大于3%,成虫与幼虫、卵无明显差异。以此能够根据COⅡ序列差异判断两个个体是否同种。然而,对于亲缘关系非常接近的铜绿蝇和丝光绿蝇来说,由于二者的种内、种间进化分歧均数非常接近,运用上述两个片段则很难区别。结论m tDNA上COⅡ序列分析能有效地对绝大多数嗜尸性苍蝇进行种类鉴定。该检测方法快速、简便和精确,能作为法医鉴别嗜尸性苍蝇种类的依据。     

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。     

基于DnaSP软件分析嗜尸性丽蝇COⅠ基因序列多态性

目的 基于DnaSP软件分析嗜尸性丽蝇线粒体细胞色素c氧化酶亚基Ⅰ（Cytochrome c Oxidase SubunitⅠ,COⅠ）基因序列,以期发现潜在的小片段多态性区域,为嗜尸性蝇类尤其是降解蝇类样本的检测以及相关技术的应用提供基础数据。方法 通过NCBI数据库检索世界范围内的嗜尸性丽蝇COⅠ序列,以丽蝇科蝇类的拉丁学名和COⅠ为关键词进行索引,序列长度大于1 300 bp为限定条件。利用MEGA7.0软件比对并进行聚类,DnaSP 5.10软件分析序列多态性。结果 获得18种常见丽蝇的224条COⅠ序列,比对剪切后终序列长度为1 206bp,存在411个可变位点,滑动窗口分析结果显示,6处可变位点数目较多,分别位于185～269 bp、290～374 bp、570～689 bp、815～934 bp、955～1 039 bp、1 060～1 179 bp。结论 长度为1 206 bp的COⅠ序列中,发现6处多态性信息丰富的小片段,这为降解蝇类样本的检测以及短片段检测技术用于嗜尸性蝇类种属鉴定提供了基础资料。     

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。     

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,...     

NCBI数据库在常见嗜尸性蝇类种属鉴别中的应用

目的探讨NCBI数据库比对分析对常见嗜尸性蝇类种属鉴别中的应用价值。方法收集2009年1月至2012年12月重庆市常见嗜尸性蝇类不同发育历期2科5属7种样本52份,采用Chelexl00法提取mtDNA,利用2对引物扩增细胞色素C氧化酶辅酶I基因,分别截取498bp和841bp相同长度的序列,采用MEGA软件计算种内及种间进化分歧情况,并分别在NCBI数据库进行序列BLAST搜索种属同源性比对分析。结果所得序列种内进化分歧均数在0％～0．7％之间,种问进化分歧均数在7．5％～16．1％之间;7个种属的样本序列Ⅰ和序列Ⅱ分别有5个和6个种属完全比对正确,样本总体的正确率分别达到96．15％和98．08％,Maxident值均在97％以上。结论采用序列同源性比对分析,并借助NCBI数据库强大的检索分析功能,可准确进行常见嗜尸性蝇类的种属鉴定,为法医学死亡时间推断提供重要参考依据。     

丝光绿蝇的发育速度和有效积温的实验观察及其法医学应用

根据丝光绿蝇在自然条件下的发育速度及其发育过程中的有效积温推断尸体的死亡时间。对在尸体腐败过程中最常见、国内分布极广的丝光绿蝇在自然条件下的发育速度及其发育过程中的有效积温进行观察、记录结果和统计学处理 ,分别确立了K1=2 0 880 + 0 80 14X1、K2 =5 4 0 917-2 8814X2 、K3=133 2 180 -2 6 312X3 等 3个有效积温与长度 (蛆 )和卵重的线性回归方程。应用丝光绿蝇发育速度与有效积温的回归方程 ,能比较准确地进行死亡时间的推断     

常见嗜尸性甲虫及蝇类虫体不同部位mtDNA的提取效果

目的探讨改良后十六烷基三甲基溴化铵(cetyltriethylammnonium bromide,CTAB)法对常见嗜尸性昆虫虫体不同部位mtDNA的提取效果。方法随机采集放置在呼和浩特地区室外草地家兔尸体上丽蝇科的丝光绿蝇[Lucilia sericata(Meigen)]、埋葬甲科的Nicrophorus fossor(Erichson)各13例,采用改良CTAB法分别提取每只昆虫头部、胸肌、腿部、翅膀4个部位的mtDNA,核酸蛋白测定仪检测DNA纯度及质量浓度,琼脂糖凝胶电泳检测其PCR产物,测定PCR产物序列,上传GenBank。结果 13例丝光绿蝇[Lu-cilia sericata(Meigen)]的胸肌均提取到了DNA,10例头部样本、6例腿部样本、4例翅膀样本得到DNA;13例Nicrophorus fossor(Erichson)胸肌DNA均提取成功,5例头部样本、8例腿部样本及3例翅膀样本提取出DNA。结论改良CTAB法可用于对嗜尸性昆虫胸肌及其他部位进行DNA的提取。     

Changes in the morphology and presumptive chemistry of impact and pooled bloodstain patterns by Lucilia sericata (Meigen) (Diptera: Calliphoridae)

Bloodstain pattern analysis can be critical to accurate crime scene reconstruction. However, bloodstain patterns can be altered in the presence of insects and can confound crime scene reconstruction. To address this problem, we conducted a series of controlled laboratory experiments to investigate the effect of Lucilia sericata (Meigen) on impact bloodstains and pooled bloodstains in association with three combinations of common surfaces (linoleum/painted drywall, wood floor/wallpaper, and carpet/wood paneling). L. sericata fed from the pooled bloodstains and added insect stains through regurgitation and defecation of consumed blood. L. sericata formed defecatory trails of insect stains that indicated directionality. Defecatory stains fluoresced when viewed at 465 nm with an orange filter. These observations differed from Calliphora vicina insect stains because feeding on blood spatter was not observed and trails of insect stains were formed by L. sericata. The fluorescence of defecatory stains can be used as a method to detect insect stains and discriminate them from real bloodstains.     

Partial sequencing of the cytochrome oxydase b subunit gene I: a tool for the identification of European species of blow flies for postmortem interval estimation

The identification of insects found on a dead body can lead to the estimation of the time of death (postmortem interval). We report an updated version of an established method based on sequence analysis of PCR products from a region of the cytochrome b oxidase subunit I mitochondrial gene of different members of the family Calliphoridae, by sequencing six European species: Lucilia sericata (Meigen), Lucilia caesar (Linné), Lucilia illustris (Meigen), Calliphora vicina (Robineau-Desvody), Calliphora vomitoria (Linné), Protophormia terraenovae (Robineau-Desvody) and one Guianese species: Cochliomyia macellaria (Fabricius). This technique provided clear results when applied to the larvae and we also report the identification of empty puparia.     

Use of PCR-RFLP for differentiation of calliphorid larvae (Diptera,Calliphoridae) on human corpses

Blowfly larvae found on human corpses are important for the estimation of the postmortem interval (PMI) and other questions of forensic relevance. Some of these species are difficult to differentiate morphologically, therefore a molecular method was elaborated for species identification. Specific fragments of the COI and COII region of the mitochondrial DNA (mtDNA) were amplified followed by digestion with different restriction enzymes. Using a 1.3 kb fragment, identification of Lucilia sericata, Calliphora vicina and Calliphora vomitoria was possible by digestion with only one restriction enzyme using either DraI or HinfI. Furthermore, we sequenced 349 bp (a part of the COI and COII regions) from the same three species and found 34 nucleotide distinctions between C. vicina and L. sericata, 30 between C. vomitoria and L. sericata and 15 between the two Calliphora species. These results aid in quick identification of species used for estimation of PMI.     

Effects of morphine in decomposing bodies on the development of Lucilia sericata (Diptera: Calliphoridae)

This study concerns the effects of morphine in tissues on the rate of development of Lucilia sericata (Diptera: Calliphoridae) using those tissues as a food source. Lucilia sericata is a species of fly commonly found on human corpses in Europe during the early stages of decomposition and thus of forensic interest. Three rabbits were administered 12.5, 25.0 and 50.0 mg/h of morphine chlorhydrate via ear perfusion over a period of 3 h. These dosages and duration of perfusion were calculated to give tissue concentrations of morphine similar to those encountered in fatal human overdoses. A fourth rabbit was used as a control. Following administration of the drug, rabbits were sacrificed and 400 eggs of Lucilia sericata, all of the same age, were placed in the eyes, nostrils and mouth of each rabbit. Developing larvae were sampled daily to determine growth rate and weight. Puparia and emerging adult flies were also sampled. Data were analyzed using analysis of variance (ANOVA) and Student's T-test. Results of this study show that an underestimation of the postmortem interval of 24 h is possible if the presence of morphine in tissues is not considered. This study demonstrates again the necessity of considering the possible effects of drugs in tissues on insect growth rates when estimating the postmortem interval using entomological techniques.     

Lucilia silvarum Meigen, 1826 (Diptera: Calliphoridae)-A new species of interest for forensic entomology in Europe

In Europe, the blowfly genus Lucilia is represented in Forensic Entomology mainly by the species L. ampullacea, L. caesar, L. illustris and L. sericata. In the US, Lucilia silvarum is rarely recorded as a carrion breeding species but usually as a more or less exclusive parasite of frogs and toads. We present three forensic cases from different European countries reporting, for the first time, L. silvarum on human bodies that were found close to lakes, wetlands, or riversides. To use this species for post-mortem interval estimations, thermal development data is needed. The first step is accurate identification by morphological and molecular means. Therefore, we analysed a 611bp part of the mitochondrial COI region for 23 specimens of L. silvarum from 9 different geographical regions, all of which give the same haplotype. Differences within the haplotype varied by up to 0.2%. Comparison between the haplotype found and those published on GenBank showed up to 1.2% variance. Moreover, we present an updated key for the morphological identification of the third larval instars of European Lucilia spp. of forensic importance, adding not only L. silvarum, but also L. cuprina which was recorded in Europe for the first time about 20 years ago.     

The application of mitochondrial DNA cytochrome oxidase II gene for the identification of forensically important blowflies in Western China

Blowflies found on human corpses are important for the estimation of the postmortem interval and other questions of forensic relevance. Some of these species are difficult to differentiate morphologically, and therefore a molecular method was elaborated for species identification. Here, we describe a molecular method for rapid identification of these insects. Specific insect DNA fragments were amplified using the polymerase chain reaction, followed by direct DNA sequencing of the amplification products. Analysis of the cytochrome oxidase II sequences revealed abundant phylogenetically informative nucleotide substitutions that could identify blowfly species to species group. In contrast, because of the low level of sequence divergence of sister species, the data could not distinguish among taxa from the same species group, ie, the species within the Lucilia sericata and Lucilia cuprina groups. The molecular data support the existing species group separation of the taxa within the Calliphora. Because of the speed and accuracy of current nucleotide sequencing technology and the abundant apomorphic substitutions available from mtDNA sequences, this approach enables quick identification of species used for estimation of postmortem interval.     

Use of Lucilia species for forensic investigations in Southern Europe

The aim of this study was to highlight the importance of evaluating entomological evidence in forensic investigations on a regional scale. To evaluate climatic, geographical and environmental influences on the selection of carrion-breeding fauna in Northern Italy and consequently on inferred forensic data (post-mortem intervals and post-mortem transfer), we present details of six indoor-outdoor cases. Results show that the most abundant species was Lucilia sericata, together with other fly species of entomo-forensic interest, belonging to the Calliphoridae and Sarcophagidae families. In particular, for the first time in Italy, we report finding Phormia regina, Lucilia ampullacea, Lucilia caesar and Sarcophaga (Pandelleana) protuberans on fresh cadavers. The active period of L. sericata in Northern Italy, according to previous findings in Southern Europe, revealing clearcut differences with phenologies in Northern Europe, has important consequences in estimating the period (season, months) of death in cases of long post-mortem intervals (several months or years) if empty puparia of this fly are found. According to our results, the distribution of L. sericata in areas with urban sprawl, like Northern Italian regions, cannot be used to evaluate post-mortem transfer from an urban area to a rural one.     

呼和浩特地区夏秋季常见嗜尸性蝇类的研究

目的研究呼和浩特地区常见嗜尸性苍蝇种类及其出现规律。方法观察法研究7～10月份兔尸上嗜尸性苍蝇种类变化。结果该地区兔尸上出现的双翅目嗜尸性苍蝇3科8属10种,主要有蝇科的家蝇、舍蝇、开普黑蝇、刺足齿股蝇、厩腐蝇;丽蝇科的丝光绿蝇、大头金蝇、铜绿蝇;麻蝇科的棕尾别麻蝇、肥须亚麻蝇。上述蝇类在尸体上出现时间及部位表现出很强的规律性。结论研究结果有望成为法医在呼和浩特地区对尸体进行死亡时间推断的依据。