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.     

Human and insect mitochondrial DNA analysis from maggots

During the course of our forensic investigations, we have encountered situations where it would have been useful to have evidence, other than direct contact between the two, for concluding that a carrion-fly maggot developed on a particular human victim. If a maggot collected during a death investigation did not develop on the victim, then its age is not relevant to estimating the postmortem interval. In this study we demonstrate that mitochondrial DNA (mtDNA) sequence data can be obtained from the dissected gut of a maggot that had fed on human tissue. These data can be used to identify both the human corpse upon which the maggot had been feeding and the species of the maggot itself.     

STR typing of human DNA from fly larvae fed on decomposing bodies

In homicides with entomological evidence, it may be important to prove the presumed association of fly larvae to a corpse, especially if it is in doubt whether all maggots used for entomological expertise developed and fed on it. The present study demonstrates for the first time the possibility of analyzing human microsatellite DNA present in the digestive tract of necrophagous larvae that fed on decomposed bodies with a postmortem interval up to four months. The obtained human STR profiles support the association of a maggot to a specific corpse. In addition, the identification of the host species (e.g., animal source like pig) can be achieved by analysis of the cytochrome b gene. Maggots were collected from 13 corpses after various postmortem intervals and STR typing and HVR amplifications were performed using their crop contents. In seven cases, a complete STR profile was established, in two cases, an incomplete set of alleles was obtained, and in four cases, STR typing was not successful. HVR analysis was successful in all cases except one. The time of storage of the maggots and the length of the postmortem interval up to 16 weeks appeared to have no particular influence on the quality of the results.     

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。     

Real-time PCR designs to estimate nuclear and mitochondrial DNA copy number in forensic and ancient DNA studies

We explore different designs to estimate both nuclear and mitochondrial human DNA (mtDNA) content based on the detection of the 5' nuclease activity of the Taq DNA polymerase using fluorogenic probes and a real-time quantitative PCR detection system. Human mtDNA quantification was accomplished by monitoring the real-time progress of the PCR-amplification of two different fragment sizes (113 and 287 bp) within the hypervariable region I (HV1) of the mtDNA control region, using two fluorogenic probes to specifically determine the mtDNA copy of each fragment size category. This mtDNA real-time PCR design has been used to assess the mtDNA preservation (copy number and degradation state) of DNA samples retrieved from 500 to 1500 years old human remains that showed low copy number and highly degraded mtDNA. The quantification of nuclear DNA was achieved by real-time PCR of a segment of the X-Y homologous amelogenin (AMG) gene that allowed the simultaneous estimation of a Y-specific fragment (AMGY: 112 bp) and a X-specific fragment (AMGX: 106 bp) making possible not only haploid or diploid DNA quantitation but also sex determination. The AMG real-time PCR design has been used to quantify a set of 57 DNA samples from 4-5 years old forensic bone remains with improved sensitivity compared with the slot-blot hybridization method. The potential utility of this technology to improve the quality of some PCR-based forensic and ancient DNA studies (microsatellite typing and mtDNA sequencing) is discussed.     

Forensic genetic analysis of insect gut contents

Entomological evidence is most often used for estimating the postmortem interval, but fly larvae can also be a source of vertebrate DNA. Forensic analysis of DNA recovered from a larva's gut can be used to identify what the larva had been feeding on. During our previous research studies, we used the same DNA extraction for the dual purpose of identifying the insect species and associating a maggot with its last meal. In our experience, we have encountered several situations where this method for associating a maggot with a corpse would have been useful, such as removal of remains from a suspected crime scene, an alternative food source is nearby the scene or the body, and a chain-of-evidence dispute. However, since maggot gut content analysis is a quite brand-new area of study, many of the limitations of the technique have not yet been explored. The results of our most recent research studies suggest that third-instar larvae actively feeding on the corpse can be considered the best source of human DNA, better than postfeeding or starved larvae. In this paper, the state of the art of forensic genetic analysis of maggot gut contents is reviewed.     

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。     

Forensic applications of the sequencing of mitochondrial DNA cytochrome oxidase subunit I gene for sarcosaphagous flies (Diptera) in Huhhot and Dunhuang district

OBJECTIVE: To solve the problems of identification of Sarcosaphagous flies and their larvae, pupas and eggs. METHODS: Sarcosaphagous Flies (Diptera) Samples were collected on the corpses of rabbits in the Huhhot district and a pig in the Dunhuang district. A 278bp region in the cytochrome oxidase subunit I (CO I) gene in mtDNA was analysed by DNA sequencing, A neighbour-joining tree using the Tamura and Nei model of nucleotide substitution was also constructed using the MEGA2.1 package. RESULTS: A 278 base pairs region of the gene for CO I encoding region of mtDNA of above all samples was showed less than 1% sequence divergence within species and about 3% divergence between species. CONCLUSION: It is an effective, easy and accurate method to be used for identification of these Sarcosaphagous Flies (Diptera) to species group by sequencing the 278 base pairs region of the CO I encoding gene of mtDNA.     

An analytical evaluation of eight on-site oral fluid drug screening devices using laboratory confirmation results from oral fluid

A decaying cadaver emits volatile organic compounds that are used by necrophilous and necrophagous insects in order to find their brood substrate. Although volatile organic compounds (VOCs) that are released by carcasses have been identified, little is known about the specific compounds that are used by these insects while searching for a brood substrate. Therefore, we have investigated the chemical ecology involved in the attraction of the necrophagous hide beetle Dermestes maculatus, which feeds as an adult and larva upon decomposing carcasses. Our aims have been to identify the responsible compounds in the odours of the carcass that are important for the attraction of the beetles. Furthermore, we have studied sex- and age-related differences in beetle attraction and tested whether the hide beetle can distinguish between various stages of decomposition by means of the emitted odours. Headspace collection of volatiles released from piglet carcasses (bloated stage, post-bloating stage, advanced decay and dry remains), coupled gas chromatography-mass spectrometry (GC-MS), gas chromatography with electroantennographic detection (GC-EAD) and bioassays were conducted to identify the volatiles responsible for the attraction of the beetles. Freshly emerged male beetles were attracted by the odour of piglets in the post-bloating stage (9 days after death; T(mean) = 27 °C) and the EAD-active compound benzyl butyrate. Statistical analysis revealed a higher relative proportion of benzyl butyrate in the odour bouquet of the post-bloating stage in comparison with the other stages. We therefore conclude that this compound plays an important role in the attraction of hide beetles to carcass odour. This underlines the potential use of D. maculatus for the estimation of the post mortem interval. The decomposition stage at which the female beetles are attracted to the odour of a cadaver remains unknown, as does the nature of this attraction. Pheromones (sexual or aggregation pheromones) might play an essential role correlated with their attraction to carrion and consequently with their attraction to the substrate for mating and ovipositioning.     

Personal identification from human remains by mitochondrial DNA sequencing

The authors report four cases in which severely damaged human remains were identified by mitochondrial DNA (mtDNA) sequencing. Degraded DNA was extracted from highly adipoceratous tissues using the phenol-chloroform method and polymerase chain reaction amplified for sequencing of two hypervariable regions, hypervariable region 1 and hypervariable region 2, of mitochondrial DNA. They also sequenced these regions of blood samples that were obtained from the presumptive mother or sister of the human remains. The sequencing results were compared with each other and with the Anderson's sequence. It was concluded from the sequence data that a lower part of a body in case 1 and some organs in case 2 were from the same woman, and a human head in case 3 and a female body in case 4 were from the relative of a presumptive mother and a sister, respectively.     

DNA Typing for the Identification of Old Skeletal Remains from Korean War Victims*

Abstract: The identification of missing casualties of the Korean War (1950–1953) has been performed using mitochondrial DNA (mtDNA) profiles, but recent advances in DNA extraction techniques and approaches using smaller amplicons have significantly increased the possibility of obtaining DNA profiles from highly degraded skeletal remains. Therefore, 21 skeletal remains of Korean War victims and 24 samples from biological relatives of the supposed victims were selected based on circumstantial evidence and/or mtDNA‐matching results and were analyzed to confirm the alleged relationship. Cumulative likelihood ratios were obtained from autosomal short tandem repeat, Y‐chromosomal STR, and mtDNA‐genotyping results, and mainly confirmed the alleged relationship with values over 10⁵. The present analysis emphasizes the value of mini‐ and Y‐STR systems as well as an efficient DNA extraction method in DNA testing for the identification of old skeletal remains.     

The Use of Mitochondrial DNA Single Nucleotide Polymorphisms to Assist in the Resolution of Three Challenging Forensic Cases

Abstract:  Mitochondrial DNA (mtDNA) single nucleotide polymorphisms (SNPs) in an 11-plex assay were typed in three missing person cases involving highly degraded human remains. Unlike the traditional forensic approach to analyzing mtDNA which focuses on sequencing portions of the noncoding Control Region, this assay targets discriminatory SNPs that reside principally in the coding region. In two of the cases, the SNP typing successfully excluded one of two reference families that could not be excluded on the basis of mtDNA hypervariable region sequencing alone, and resulted in the final resolution of both decades-old cases. In a third case, SNP typing confirmed the sorting and reassociation of multiple commingled skeletal elements. The application of a specific mtDNA SNP assay in these cases demonstrates its utility in distinguishing samples when the most common Caucasian hypervariable region type is encountered in forensic casework.     

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.     

Determination of drug levels in two species of necrophagous Coleoptera reared on substrates containing morphine

Two species of necrophagous Coleoptera: Dermestes frischi (Dermestidae) and Thanatophilus sinuatus (Silphidae), were reared on substrates containing different amounts of morphine. Colonies of D. frischi were reared on rabbit carcasses which had been given 10, 20, and 40 mg/h of morphine hydrochloride via ear artery perfusion over a 3 h period prior to death. A fourth rabbit served as a control. T. sinuatus was reared on minced beef spiked with morphine hydrochloride to give concentrations of 1,000, 2,500, 5,000, and 10,000 ng/g and one control colony. These dosages were calculated to create tissue concentrations of morphine similar to those encountered in human deaths due to morphine overdose. Larvae. pupae, and adults (except for T. sinuatus) were analyzed for morphine content. All developmental stages of D. frischi were positive for morphine and concentrations correlated with cadaveric tissue concentrations during larval stages and to a lesser extent in the adult stage. For T. sinuatus, the best correlations were found in 2nd and 3rd instar larvae. This study demonstrates the potential for use of necrophagous Coleoptera, as well as Diptera larvae, as alternate specimens for toxicological analyses.     

北京地区尸食性蝇类调查

目的了解北京地区目前尸食性蝇类数目及常见尸食性蝇类的季节分布。方法采用动物尸体或组织诱捕和饲养的方法采集尸食性蝇类标本,对其进行分类保存后进行观察和统计。结果北京地区尸食性蝇类为4科9亚科21属46种,其中有12种是北京新记录种。尸食性蝇类具有区域性分布和季节性分布的特性。结论北京地区尸食性蝇类及常见的尸食性蝇类季节分布数据,可为相关研究提供参考依据。     

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。     

The Correlation Between Skeletal Weathering and DNA Quality and Quantity*

Abstract: Mitochondrial DNA analysis of skeletal material is invaluable in forensic identification, although results can vary widely among remains. Previous studies have included bones of different ages, burial conditions, and even species. In the research presented, a collection of human remains that lacked major confounders such as burial age, interment style, and gross environmental conditions, while displaying a very broad range of skeletal degradation, were examined for both mitochondrial DNA (mtDNA) quality and quantity. Overall skeletal weathering, individual bone weathering, and bone variety were considered. Neither skeletal nor bone weathering influenced DNA quality or quantity, indicating that factors that degrade bone do not have the same effect on DNA. In contrast, bone variety, regardless of weathering level, was a significant element in DNA amplification success. Taken together, the results indicate that neither skeletal nor individual bone appearance are reliable indicators of subsequent mtDNA typing outcomes, while the type of bone assayed is.     

粪便DNA提取及检验

目的　研究人类粪便DNA的提取和检验方法。方法　 8人份粪便样本 ,磁珠法提取DNA后 ,进行STR复合扩增和mtDNAHVI区测序分析。结果　用 2种方法提取的粪便DNA ,STR复合扩增检验均未获成功 ;方法1提取的粪便DNA有 6个样本、方法 2有 7个样本获得了清晰可读的mtDNAHVI区序列 ,并与唾液对照样本DNA的序列完全一致。结论　用本文建立的方法提取粪便DNA ,不适于STR分析 ,可通过mtDNA测序分析进行检验。     

The effects of skeletal preparation techniques on DNA from human and non-human bone

The forensic pathologist increasingly relies on the forensic anthropologist to be the consulting expert in human identification. Likewise, if identification is not possible from visual inspection of skeletal remains, the forensic biologist may be called upon to conduct DNA analysis. The possibility of downstream DNA testing needs to be considered when skeletal preparation techniques are employed to deflesh human remains, as they have the potential to strongly impact genetic analyses and subsequent identification. In this study, three cleaning techniques, boiling bone in water, in bleach, and in powdered detergent/sodium carbonate, were tested for their effect on nuclear and mtDNA recovery from a variety of human and non-human bones. A statistically significant reduction in DNA yields occurred in non-human bones cleaned with bleach, and DNA degradation was apparent electrophoretically. The human bones also showed much lower yields from bleach cleaning, while the detergent/carbonate method allowed the largest segments of DNA to be amplified, indicating it may have a less degradative effect on bone DNA than either of the other cleaning processes.