How many DNA analyses are performed on adult sexual assault victims in Milan (Italy): A ten-year review

The aim of this study is to evaluate the actual use of serological/DNA analyses in the investigations carried out on adult sexual violence victims in Italy during the years 2006–2015.The victims were assisted in the largest Italian rape center, in Milan (Soccorso Violenza Sessuale e Domestica – SVSeD - Service for Sexual and Domestic Violence).The total number of sexual violence victims examined during the years 2006–2015 (adults and minors) was 3521, in 1697 of cases, biological evidence had been collected, while the number of adult victims (>18 y.o.) examined was 2300, in 1211 of cases biological evidence had been collected.Biological evidence was collected from the victims’ bodies using two swabs in five anogenital areas (labia maiora, labia minora, perineum, perianal and anal/rectum regions) and two swabs in all other skin areas suggested by the victims as areas of possible contact (double swab technique). Clothes were also collected on a case by case basis for the search of biological stains. Despite the proper collection, handling and chain of custody for all the swabs/items collected, serological/DNA analyses were requested in 86 cases out of 1211 only (710%). This percentage dropped to 190% when considering adolescent victims (13–19 y.o.).The reason why Italian Magistrates make little use of the powerful tool of DNA analyses in sexual assault cases, still remains unclear. Legal and procedural aspects are therefore also discussed.     

Efficiency of Casework Direct Kit for extraction of touch DNA samples obtained from cars steering wheels

Analysis of STR profiles obtained from touch DNA has been very useful to the elucidation of crimes. Extraction method may be determinant for the recovery of genetic material collected from different surfaces. Vehicle theft is one of the most common crimes in São Paulo city, Brazil, but collection of biological traces in car steering wheels is not considered, because of the belief that profiles generated won’t be able to identify the thief, only the owner. This study aimed to analyze the efficacy of extraction methods for obtaining DNA profiles in samples collected from steering wheels. Eight criminal acts were simulated with 2 different individuals each (mixture of victim and thief), in duplicate, in order to compare two extraction methods: DNA IQ™ and Casework Direct Kit (both Promega Corporation). Genetic material was collected by double swab method and quantified by Quantifiler™Trio (ThermoFisher Scientific). Amplification was conducted with PowerPlex® Fusion System (Promega). It was possible to obtain STR profiles for all experiments. The mixtures were compared with reference profiles to evaluated how many alleles of each donor were observed. Samples extracted with Casework Direct Kit obtained STR profiles with higher averages of alleles for primary and secondary donors (88.7% and 59.9%, respectively) than those extracted with DNA IQ™ (60.4% and 38.1%, respectively). This could be explained by the differences established in the protocols of both methods, since DNA IQ™ is based on successive washes and can result in loss of DNA, whereas Casework Direct Kit minimizes this problem. We concluded that Casework Direct Kit was more efficient for processing touch DNA samples than DNA IQ™.     

The Effectiveness of Trace DNA Profiling—A Comparison Between a U.S. and a U.K. Law Enforcement Jurisdiction

Recovery, profiling, and speculative searching of trace DNA (not attributable to a body fluid/cell type) over a twelve‐month period in a U.S. Crime Laboratory and U.K. police force are compared. Results show greater numbers of U.S. firearm‐related items submitted for analysis compared with the U.K., where greatest numbers were submitted from burglary or vehicle offenses. U.S. multiple recovery techniques (double swabbing) occurred mainly during laboratory examination, whereas the majority of U.K. multiple recovery techniques occurred at the scene. No statistical difference was observed for useful profiles from single or multiple recovery. Database loading of interpretable profiles was most successful for U.K. items related to burglary or vehicle offenses. Database associations (matches) represented 7.0% of all U.S. items and 13.1% of all U.K. items. The U.K. strategy for burglary and vehicle examination demonstrated that careful selection of both items and sampling techniques is crucial to obtaining the observed results.     

Inferring the Number of Contributors to Complex DNA Mixtures Using Three Methods: Exploring the Limits of Low‐Template DNA Interpretation

In forensic DNA casework, the interpretation of an evidentiary profile may be dependent upon the assumption on the number of individuals from whom the evidence arose. Three methods of inferring the number of contributors—NOCIt, maximum likelihood estimator, and maximum allele count, were evaluated using 100 test samples consisting of one to five contributors and 0.5–0.016 ng template DNA amplified with Identifiler^® Plus and PowerPlex^® 16 HS. Results indicate that NOCIt was the most accurate method of the three, requiring 0.07 ng template DNA from any one contributor to consistently estimate the true number of contributors. Additionally, NOCIt returned repeatable results for 91% of samples analyzed in quintuplicate, while 50 single‐source standards proved sufficient to calibrate the software. The data indicate that computational methods that employ a quantitative, probabilistic approach provide improved accuracy and additional pertinent information such as the uncertainty associated with the inferred number of contributors.     

基于Ion Torrent PGM~(TM)测序系统的人mtDNA全测序分析

目的应用Ion Torrent PGM~(TM)测序系统对人线粒体DNA(mitochondria DNA,mtDNA)全序列进行分析检测,研究不同组织间mt DNA序列差异情况。方法通过法医尸体检验采集6名无关个体的组织样本,包括胸腔血液、头发、肋软骨、指甲、骨骼肌和口腔上皮。使用4对引物对线粒体全序列进行扩增,应用Ion Shear~(TM)Plus Reagents试剂盒和Ion Plus Fragment Library试剂盒等构建文库,并在Ion Torrent PGM~(TM)测序系统上进行线粒体基因组全序列测序,并针对异质性位点和在HVⅠ区域突变位点,进行Sanger测序验证。结果所有样本的全基因组mtDNA都扩增成功,6名无关个体分属于6种不同的单倍型,同一个体不同组织之间mtDNA存在异质性差异。异质性位点和HVⅠ区域突变位点采用Sanger测序结果均得到验证。通过Kappa统计方法进行一致性检验后发现,相同个体不同组织的mtDNA序列检验结果仍具有较好的一致性。结论本研究所采用的人线粒体基因组全序列的测序检验方法,可以检测出同一个体不同组织间mtDNA的异质性差异,该差异具有较高的一致性,该结果对mtDNA在法庭科学中的应用具有指导作用。     

DNA条形码技术在兽医寄生虫学研究中的应用

阐述了DNA条形码的概念、DNA条形码基因的筛选方法、DNA条形码的优势与劣势和分析方法,概述了近几年DNA条形码在兽医寄生虫方面的应用情况,旨在引起广大读者对该技术的关注。     

Identification of forensically important sarcophagid flies (Diptera: Sarcophagidae) in China, based on COI and 16S rDNA gene sequences

Insects attracted to cadavers may provide important indications of the postmortem interval (PMI). However, use of the flesh flies (Diptera: Sarcophagidae) for PMI estimation is limited as the species are often not morphologically distinct, especially as immatures. In this study, 23 forensically important flesh flies were collected from 13 locations in 10 Chinese provinces. Then, a 278-bp segment of the cytochrome oxidase subunits one (COI) gene and a 289-bp segment of the 16S rDNA gene of all specimens were successfully sequenced. Phylogenetic analysis of the sequenced segments showed that all sarcophagid specimens were properly assigned into four species (Boerttcherisca peregrina [Robineau-Desvoidy, 1830], Helicophagella melanura [Meigen, 1826], Parasarcophaga albiceps [Meigen, 1826], and Parasarcophaga dux [Thompson, 1869]) with relatively strong supporting values, thus indicating that the COI and 16S rDNA regions are suitable for identification of sarcophagid species. The difference between intraspecific threshold and interspecific divergence confirmed the potential of the two regions for sarcophagid species identification.     

Quantification of human mitochondrial DNA using synthesized DNA standards

Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real-time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay's 105-base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust.     

Field testing of collection cards for Cannabis sativa samples with a single hexanucleotide DNA marker

The validity and feasibility of using DNA collection cards in the field for preservation and analysis of Cannabis sativa genotypes were investigated using a highly specific hexanucleotide marker. Collection cards were submitted to the National Marijuana Initiative, which selectively trained and managed the collection of specific types of samples from a variety of participating agencies. Samples collected at seizure sites included fresh marijuana leaf samples, dried "dispensary" samples, U.S. border seizures, and hashish. Using a standardized PCR kit with custom-labeled oligonucleotide primers specific to marijuana, collection cards produced eight genotypes and 13 different alleles, extremely low baselines, and no cross-reactivity with control plant species. Results were produced from all sample types with the exception of hashish. Plant DNA collection cards represent an easily implementable method for the genetic identification and relatedness of C. sativa street and grow site-seized samples with applications for databasing and market disruption.