mtDNA多态性在嗜尸性昆虫种类鉴定中的应用

嗜尸性昆虫的种类鉴定是利用昆虫学知识进行法医学研究的重要步骤之一.近年来研究表明,利用分子生物学技术,特别是线粒体DNA(mitochondrial DNA,mtDNA)序列分析技术对嗜尸性昆虫进行种类鉴别,能够得到较好的效果.本文对嗜尸性昆虫mtDNA的分子生物学特性、结构以及嗜尸性昆虫种类鉴定中常用的mtDNA序列...     

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.     

The last Viking King: a royal maternity case solved by ancient DNA analysis

The last of the Danish Viking Kings, Sven Estridsen, died in a.d. 1074 and is entombed in Roskilde Cathedral with other Danish kings and queens. Sven's mother, Estrid, is entombed in a pillar across the chancel. However, while there is no reasonable doubt about the identity of Sven, there have been doubts among historians whether the woman entombed was indeed Estrid. To shed light on this problem, we have extracted and analysed mitochondrial DNA (mtDNA) from pulp of teeth from each of the two royals. Four overlapping DNA-fragments covering about 400bp of hypervariable region 1 (HVR-1) of the D-loop were PCR amplified, cloned and a number of clones with each segment were sequenced. Also a segment containing the H/non-H specific nucleotide 7028 was sequenced. Consensus sequences were determined and D-loop results were replicated in an independent laboratory. This allowed the assignment of King Sven Estridsen to haplogroup H; Estrid's sequence differed from that of Sven at two positions in HVR-1, 16093T-->C and 16304T-->C, indicating that she belongs to subgroup H5a. Given the maternal inheritance of mtDNA, offspring will have the same mtDNA sequence as their mother with the exception of rare cases where the sequence has been altered by a germ line mutation. Therefore, the observation of two sequence differences makes it highly unlikely that the entombed woman was the mother of Sven. In addition, physical examination of the skeleton and the teeth strongly indicated that this woman was much younger (approximately 35 years) at the time of death than the 70 years history records tell. Although the entombed woman cannot be the Estrid, she may well be one of Sven's two daughters-in-law who were also called Estrid and who both became queens.     

Binge drinking and negative alcohol-related behaviors: A test of self-control theory

Binge drinking and alcohol-related behaviors have been viewed as major concerns on college campuses. Although national studies were conducted to describe these behaviors, less research attempted to explain them. Self-control theory is extended as a theoretical framework to explain both while considering other known risk factors. Using a sample of college students (n = 268) from a university in the southern United States, the additive and interactive effects of self-control were modeled to predict binge drinking and negative alcohol-related behaviors. A series of multivariate regression models showed that low self-control had effects on binge drinking and related behaviors. Binge drinking's effect on negative alcohol-related behaviors varied across levels of self-control.     

Mitochondrial DNA sequencing of beetle larvae (Nitidulidae: Omosita) recovered from human bone

The isolation, amplification, and characterization of human DNA from hematophagous (blood feeding) and necrophagous (carrion feeding) arthropods have been advanced significantly by the development of polymerase chain reaction (PCR) DNA sequencing methodologies. Historically, DNA technology has been successfully utilized to identify individual hosts upon which species of hematophagous arthropods have fed. The analysis of hematophagous insects' gut content blood meals has led to major advances in medical entomology and vector-borne disease epidemiology. In the forensic arena, the ability to apply similar techniques to insects recovered from badly decomposed remains has been greatly enhanced through the advent of mitochondrial DNA (mtDNA) techniques. Mitochondrial DNA analyses have been utilized to identify both the human remains upon which fly larvae (maggots) have fed and the species of the larvae themselves. The preliminary work detailed here demonstrates, for the first time, the successful application of mtDNA sequencing techniques to the analysis of necrophagous beetle larvae. A small sample of sap beetle larvae, Omosita spp. (Coleoptera: Nitidulidae), was collected from human skeletal remains during anthropological examination and analyzed for human DNA using mtDNA sequencing. The beetle larvae yielded mtDNA matching that of the host human bone. The results detailed here further demonstrate the robust nature of human mtDNA and the ability to recover valuable mtDNA evidence from forensically important, late decompositional stage insect species.     

A simple and inexpensive molecular method for sexing and identification of the forensic samples of elephant origin

The population of the Asian elephant is being dramatically reduced due to poaching of the ivory from the male. As poaching occurs in remote forests, it often takes weeks or longer for it to be discovered and it is therefore often very difficult to determine the sex of the decomposed body. Data suggest that in the recent past, over 2000 male elephants have been poached in South India. We have developed a technique based on molecular markers to determine that the carcass is an elephant and that it is a male. Using DNA sequence information from Genbank, we have developed two primer pairs: one for the mitochondrial DNA (mtDNA) and the other for the sex-determining region of Y chromosome (SRY) gene of the Indian elephant. After PCR amplification of known elephant DNA, we found that the mtDNA was common in both males and females, whereas the SRY-specific amplicon was observed only in the male.     

A global study of forensically significant calliphorids: implications for identification

A proliferation of molecular studies of the forensically significant Calliphoridae in the last decade has seen molecule-based identification of immature and damaged specimens become a routine complement to traditional morphological identification as a preliminary to the accurate estimation of post-mortem intervals (PMI), which depends on the use of species-specific developmental data. Published molecular studies have tended to focus on generating data for geographically localised communities of species of importance, which has limited the consideration of intraspecific variation in species of global distribution. This study used phylogenetic analysis to assess the species status of 27 forensically important calliphorid species based on 1167 base pairs of the COI gene of 119 specimens from 22 countries, and confirmed the utility of the COI gene in identifying most species. The species Lucilia cuprina, Chrysomya megacephala, Ch. saffranea, Ch. albifrontalis and Calliphora stygia were unable to be monophyletically resolved based on these data. Identification of phylogenetically young species will require a faster-evolving molecular marker, but most species could be unambiguously characterised by sampling relatively few conspecific individuals if they were from distant localities. Intraspecific geographical variation was observed within Ch. rufifacies and L. cuprina, and is discussed with reference to unrecognised species.     

Cytochrome b based genetic differentiation of Indian wild pig (Sus scrofa cristatus) and domestic pig (Sus scrofa domestica) and its use in wildlife forensics

The Indian wild pig (Sus scrofa cristatus) is a protected species and listed in the Indian Wildlife (Protection) Act, 1972. The wild pig is often hunted illegally and sold in market as meat warranting punishment under law. To avoid confusion in identification of these two subspecies during wildlife forensic examinations, we describe genetic differentiation of Indian wild and domestic pigs using a molecular technique. Analysis of sequence generated from the partial fragment (421 bp) of mitochondrial DNA (mtDNA) cytochrome b (Cyt b) gene exhibited unambiguous (> 3%) genetic variation between Indian wild and domestic pigs. We observed nine forensically informative nucleotide sequence (FINS) variations between Indian wild and domestic pigs. The overall genetic variation described in this study is helpful in forensic identification of the biological samples of wild and domestic pigs. It also helped in differentiating the Indian wild pig from other wild pig races. This study indicates that domestic pigs in India are not descendent of the Indian wild pig, however; they are closer to the other wild pig races found in Asia and Europe.     

A Checklist of Calliphoridae Blowflies (Insecta,Diptera) Associated with a Pig Carrion in Central Brazil*

Abstract: Although the Cerrado is the second major Brazilian biome, few studies have been undertaken about its entomofauna. Blowflies have an important role in forensic entomology, helping in the determination of postmortem intervals. The main goal of this exploratory study was to identify and to catalog the blowfly species associated with a pig carcass. The study was conducted in a pasture in Brasília, Distrito Federal. A pig (Sus scrofa) was killed with a .22 caliber shot in the frontal region of the head. Adult blowflies were surveyed daily from June 1 through 30, 2004. A total of 14,910 adult calliphorids were collected, representing eight species: Chrysomya albiceps, C. megacephala, Cochliomyia macellaria, Chloroprocta idioidea, Hemilucilia semidiaphana, H. segmentaria, Lucilia cuprina, and L. eximia. C. albiceps was the most frequent species, amounting to 94.76% of the catch. Five decomposition stages were observed, and for calliphorids, the most attractive stage was the bloated one.     

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.     

Rural and urban differences in the commission of animal cruelty

Despite the recent surge in society's interest in human violence, relatively few studies have been conducted examining the closely related phenomenon of animal cruelty. Although several researchers have begun to identify some of the correlates of animal cruelty, few have attempted to understand how differences in the backgrounds of rural and urban residents have led to their abuse of animals. Using survey data from 261 inmates, the authors investigate how demographic, familial differences and species type have contributed to the frequency of acts of animal cruelty. In general, early exposure to animal abuse is a strong predictor of the subsequent behavior. However, rural inmates learned to be cruel by watching family members exclusively, whereas urban inmates learned from family members and friends. Moreover, urban inmates chose dogs, cats, and wild animals as their target animals; however, rural inmates chose only cats.     

Sentencing practices under the Arkansas sentencing guideline structure

In the last thirty years, research on differential sentencing practices became one of the dominant thrusts of academic interest in criminal justice studies. This was mainly because several reforms had been added to the various sentencing structures in the United States, one of which was the adoption of guideline based sentencing strategies. Although several studies were conducted regarding the impact of these guidelines, these were provided by a limited number of sources. For instance, the majority of this research was conducted on data collected by the United States Sentencing Commission and the Pennsylvania Sentencing Commission. This study attempted to further the discussion on sentencing practices by examining data from a new source, the Arkansas Office of Courts. This study first examined the judge's decision to imprison and jail the defendant using logistic regression, and second, for those individuals incarcerated, OLS and negative binomial regression analyses were conducted to explore potential disparities in the length of prison and jail sentences. The results of this analysis in Arkansas illustrated striking comparisons to studies that were conducted on other guideline structures. The legally relevant variables were the greatest predictors of whether or not an offender was sentenced to prison. Extralegal variables, on the other hand, had negligible effects. Implications for policy are also discussed.     

Key longitudinal-experimental studies in criminology

Following the tradition of Joan McCord's work, this paper reviews longitudinal-experimental studies in criminology with community samples of at least 100 persons, follow-up periods of at least 5 years, personal interviews, and measures of offending. The main advantages of such studies are in investigating both the natural history of development (including the effects of risk/protective factors and life events) and the impact of interventions on offending. This paper also reviews advantages and problems of prospective longitudinal surveys, randomized experiments, and longitudinal-experimental studies in criminology. Four key longitudinal-experimental studies were conducted by Joan McCord, Richard Tremblay, Lawrence Schweinhart and David Olds. Other studies have been conducted, or are currently under way, but no criminological study has yet been published with several years of personal data on participants both before and after an intervention.Joan McCord Award Lecture given at the American Society of Criminology, Toronto, Canada.     

Mitochondrial DNA and STR typing of matter adhering to an earphone

STR typing and mitochondrial DNA (mtDNA) sequencing were performed on the matter adhering to an earphone found at a crime scene. Experimental studies were carried out using the earphones provided by volunteers. By means of immunohistochemistry, keratinocytes and a portion of nucleated epithelial cells were proven to exist in the contents from the earphones. DNA was extracted by means of the phenol/chloroform method, and the low quantity of extracted DNA was found to be highly degraded. Six STR loci, CSFIPO, TPOX, TH01, F13A01, FESFPS and vWA, were PCR amplified and typed by using two triplex systems (CTT and FFv Multiplexes, Promega, WI), and an amelogenin locus was determined as well. Although partial profiles were observed in some experimental samples, all STR loci could be typed when a considerable amount of high molecular weight DNA was obtained (>0.5 ng/microL). Amplification and sequencing of mtDNA hypervariable region I(15997-16401) and hypervariable region 11(29-408) were all successful. The mitochondrial DNA sequence of the actual case sample, comprising two hypervariable regions and a total of 785 base pairs, showed eight mutations and two insertions with respect to the standard published reference sequence. The genotype was unique in the three published Japanese databases. These results suggest that it is possible to analyze mtDNA from minute amounts of materials and from degraded materials more effectively and routinely in forensic practice.     

A METHODOLOGICAL ADDITION TO THE CROSS‐NATIONAL EMPIRICAL LITERATURE ON SOCIAL STRUCTURE AND HOMICIDE: A FIRST TEST OF THE POVERTY‐HOMICIDE THESIS*

Dozens of cross‐national studies of homicide have been published in the last three decades. Although nearly all these studies test for an association between inequality and homicide, no studies test for a poverty—homicide association. This absence is disconcerting given that poverty is one of the most consistent predictors of area homicide rates in the abundant empirical literature on social structure and homicide in the United States. Using a sample that coincides closely with similar recent studies, applying a proxy for poverty (infant mortality) that is commonly employed in noncriminological cross‐national research, and controlling for several common covariates (including inequality), this study provides the first test of the poverty—homicide hypothesis at the cross‐national level. The results reveal a positive and significant association between a nation's level of poverty and its homicide rate. The findings also suggest that we may need to reassess the strong conclusions about an inequality—homicide association drawn from prior studies, as this relationship disappears when poverty is included in the model.     

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.     

Quantification of trace amounts of human and non-human mitochondrial DNA (mtDNA) using SYBR Green and real time PCR

There are currently no tests available to quantify total non-human mammalian mtDNA. Standard universal DNA quantification tests are unsuitable due to the large size difference between nuclear and mitochondrial genomes and the ubiquity of human mtDNA. A method has therefore been developed to quantify total mammalian mtDNA and total human mtDNA present in a sample using SYBR Green.Mammalian primers designed to react with all mammals were designed on the 12S and human specific primers were designed on the cytochrome b gene. Each primer set was reacted separately with sample and SYBR Green and detected using RT-PCR. A standard curve was developed using dilutions ranging from 1 billion copies to 100 copies of mtDNA.Twenty-four human samples were analysed and an average log (copy number) human/universal ratio of 1.00 was obtained. Samples falling below this ratio will contain some non-human mtDNA while samples falling above this ratio contain human mtDNA only.Twenty-nine mammal samples were also tested. 96.6% of these showed human contamination to some extent. This test is able to quantify mtDNA down to the femtogramme (10E−15g) level.     

Current Next Generation Sequencing technology may not meet forensic standards

In a Nature paper of 2010, the concern was raised that intra-individual mtDNA variation may be more pronounced than previously believed, in that heteroplasmies are common and vary markedly from tissue to tissue. This claim taken at face value would have considerable impact on forensic casework. It turns out however that the employed technology detected the germ-line variation relative to the reference sequence only incompletely: on average at least five mutations were missed per sample, as an in silico reassessment of the data reveals. Before one can really set out to access to entire mtDNA genome data with relative ease for forensic purposes, one needs careful calibration studies under strict forensic conditions—or might have to wait for another generation.     

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.