DNA barcoding as a tool for species identification in three forensic wildlife cases in South Africa

Poaching of wildlife animals for subsistence and commercial purposes has lead to population declines in Africa. In forensic cases, a need exists to identify the species of origin of carcasses, meat or blood. In the study presented here, the mitochondrial COI gene was sequenced to determine the species of unknown samples in three suspect South African forensic wildlife cases. In two cases the unknown samples were identified as originating from domestic cattle (Bos taurus) and in the third case the sample was identified as common reedbuck (Redunca arundinum). This is the first report of the COI sequence of common reedbuck. The study highlights the need for accurate wildlife reference material from each country in order to convict wildlife cases.     

Integration of the AmpFlSTR Identifiler PCR Amplification Kit with SRY-specific primers for gender identification

Dropout of the amelogenin Y-specific allele due to an interstitial deletion of the Yp involving the amelogenin Y locus (AMELY) can cause misidentification of sex genotype with potentially serious consequences in personal identification processes and criminal investigations. Inclusion of additional sex-defining markers in forensic DNA typing kits is therefore advisable. In this study, the co-amplification of the sex-determining region Y (SRY) gene and 16 STR loci included in the AmpFlSTR Identifiler PCR Amplification Kit was evaluated. Combination of SRY and Identifiler primers did not compromise the amplification outcome and generated a 90 bp male-specific SRY fragment, showing a reproducible peak height ratio in comparison with the AMELY peak. The SRY peak was detectable in presence of amounts of template DNA as low as 125 pg, and in mixed samples with a male/female DNA ratio of 1:100.     

Molecular Assay for Screening and Quantifying DNA in Biological Evidence: The Modified Q‐TAT Assay*

Abstract: A method is described for the quantitation of total human and male DNA. Q‐TAT utilizes end‐point, multiplex polymerase chain reaction (PCR) amplification of the amelogenin and SRY loci to quantify DNA and incorporates a cloned nonhuman template to detect PCR inhibition. Standard curves of fluorescence from amelogenin or SRY amplicons were generated from amplification of known amounts of NIST traceable SRM‐female or SRM‐male DNA. Curves showed good linearity up to 500 pg of SRM‐template (R² > 0.99) and reliably estimated total and male DNA content in casework samples. The nonhuman pRL_null template included in each PCR was a sensitive indicator of known PCR inhibitors including EDTA, hemin, blue denim dye, and humic acid. Finally, the SRY amplicon was a sensitive indicator of male DNA and, in mixtures, could reliably estimate male DNA present in an excess of female DNA. The Q‐TAT multiplex is a reliable quantitation method for forensic DNA typing.     

DNA Barcoding Identifies all Immature Life Stages of a Forensically Important Flesh Fly (Diptera: Sarcophagidae)

Carrion‐breeding insects, such as flesh flies (Diptera: Sarcophagidae), can be used as evidence in forensic investigations. Despite their considerable forensic potential, their use has been limited because morphological species identification, at any life stage, is very challenging. This study investigated whether DNA could be extracted and cytochrome oxidase subunit I (COI) barcode sequences obtained for molecular identification of each immature life stage of the forensically important Australian flesh fly, Sarcophaga (Sarcorohdendorfia) impatiens (Walker). Genomic DNA extracts were prepared from all larval instars and puparia. Amplification of the barcoding region was successful from all extracts, but puparia amplicons were weak. All sequences were identified as S. impatiens with 99.95% confidence using the Barcoding of Life Database (BOLD). Importantly, crop removal was necessary to eliminate PCR inhibition for specimens from late second and early third instars. Similar results are expected for immatures of other carrion‐breeding species, enhancing the use of evidence from immature flies in forensic investigations.     

Analysis of 11 tetrameric STRs in wild boars for forensic purposes

STR profiling of animal species has a wide range of applications, including forensic identification, wildlife preservation, veterinary public health protection and food safety. We tested the efficacy of a multiplex PCR-based assay including 11 porcine-specific tetrameric STRs in a population sample of wild boars (n = 142) originating from Piedmont (North West Italy). Multiple deviations from Hardy–Weinberg expectations were observed, mostly due to a reduction in observed heterozygosity indicative of a high degree of inbreeding. A value of θ of 0.046 and an inbreeding coefficient of 0.089 were estimated. Combined power of discrimination and probability of exclusion values for the STR panel were 0.9999999999996 and 0.99989. In order to test the suitability of the method for meat traceability purposes, a domestic pig reference sample (n = 412), consisting of commercial lines commonly used in the meat production process, was also typed. A Bayesian cluster analysis carried out using the observed genotypes, showed a percentage of correct subspecies assignment of individual samples of 0.974 for wild boars and 0.991 for pigs, thus demonstrating the usefulness of the multiplex STR-typing system for discrimination purposes.     

Failure of anthropometry as a facial identification technique using high-quality photographs

Anthropometry can be used in certain circumstances to facilitate comparison of a photograph of a suspect with that of the potential offender from surveillance footage. Experimental research was conducted to determine whether anthropometry has a place in forensic practice in confirming the identity of a suspect from a surveillance video. We examined an existing database of photographic lineups, where one video image was compared against 10 photographs, which has previously been used in psychological research. Target (1) and test (10) photos were of high quality, although taken with a different camera. The anthropometric landmarks of right and left ectocanthions, nasion, and stomion were chosen, and proportions and angle values between these landmarks were measured to compare target with test photos. Results indicate that these measurements failed to accurately identify targets. There was also no indication that any of the landmarks made a better comparison than another. It was concluded that, for these landmarks, this method does not generate the consistent results necessary for use as evidence in a court of law.     

Meeting a Forensic Podiatry Admissibility Challenge: A Daubert Case Study

This article is an introduction to the United States Supreme Court's standard of admissibility of forensic evidence and testimony at trial, known as the Daubert standard, with emphasis on how this standard applies to the field of forensic podiatry. The author, a forensic podiatrist, provided law enforcement with evidence tying a bloody sock‐clad footprint found at the scene of a homicide to the suspect. In 2014, the author testified at a pretrial hearing, known as “a Daubert hearing,” to address the admissibility of this evidence in court. This was the first instance of forensic podiatry being the primary subject of a Daubert hearing. The hearing resulted in the court ordering this evidence admissible. The expert's testimony contributed to the suspect's conviction. This article serves as a reference for forensic podiatrists and experts in similar fields that involve impression evidence, providing evidentiary standards and their impact on expert evidence and testimony.     

Forensic DNA against wildlife poaching: Identification of a serial wolf killing in Italy

The recent expansion of the Italian wolf population through the Apennine and western Alps, after centuries of contractions, is causing conflicts with human activities leading to a rise in poaching or illegal killings. Here we show how molecular population genetics has been used to identify a suspect serial wolf killer. We analysed DNA extracted from a necklace made of ten presumed wolf canine teeth, confiscated in 2008 to a man living in the northern Italian Apennine (Liguria Region). Individual genotypes were determined using 12 unlinked autosomal microsatellites (STRs), mtDNA control-region sequences, a male-specific ZFX/ZFY restriction-site and three Y-linked STRs. Results indicate that the teeth belonged to six different individuals (three males and three females), which were assigned to the Italian wolf population with p > 0.90 by Bayesian procedures. One of these genotypes matched with the genetic profile of a male wolf previously found-dead and already non-invasively sampled in the same area. Another genotype matched with that of a female wolf non-invasively sampled twice in the same area 1 year before. These data are being used as forensic genetic evidence in the ongoing criminal trial against the suspect serial wolf killer.     

DNA forensics and the poaching of wildlife in Italy: a case study

DNA molecular techniques were used in a forensic investigation involving the poaching of wildlife in a national park of Italy. A poacher, after having snared a wild boar (Sus scrofa) sow, knifed it to death. The animal was retrieved by conservation officers at the scene before the poacher could remove the carcass. Subsequently, the suspect denied the charges. During a search of his home, a bloodstained knife was confiscated. A method to identify the species from the DNA extracted from the stains revealed the blood to be that of the non-domestic form of Sus scrofa. Further DNA typing for individual identity using species-specific single tandem repeats or microsatellites (STRs) showed that the DNA on the knife matched that of the poached boar. Based upon the forensic evidence obtained, the suspect was convicted of poaching and of cruelty to animals.     

Instruction Bias and Lineup Presentation Moderate the Effects of Administrator Knowledge on Eyewitness Identification

Pairs (N = 234) of witnesses and lineup administrators completed an identification task in which administrator knowledge, lineup presentation, instruction bias, and target presence were manipulated. Administrator knowledge had the greatest effect on identifications of the suspect for simultaneous photospreads paired with biased instructions, with single-blind administrations increasing identifications of the suspect. When biased instructions were given, single-blind administrations produced fewer foil identifications than double-blind administrations. Administrators exhibited a greater proportion of biasing behaviors during single-blind administrations than during double-blind administrations. The diagnosticity of identifications of the suspect in double-blind administrations was double their diagnosticity in single-blind administrations. These results suggest that when biasing factors are present to increase a witness’s propensity to guess, single-blind administrator behavior influences witnesses to identify the suspect.     

Age Estimation in Living Egyptians Using Signal Joint T‐cell Receptor Excision Circle Rearrangement

Age estimation is one of the challenges in forensic sciences. There are many techniques to estimate the age. Molecular biology approach is one of these techniques. Signal joint T‐cell receptor excision circles gene (sjTRECs), is one of this approach. We aimed to use sjTRECs as a suitable marker for age estimation among Egyptian population. TaqMan qPCR approach was used to quantify sjTREC levels in blood samples obtained from 153 healthy Egyptian individuals ranging from a few weeks to 70 years. Our results showed a significant negative correlation between sjTREC levels and age with p ≤ 0.05. Moreover, the individual's age can be determined through this formula Age = ?30.671+ (?5.998Y) (Y is dCtTBP ? sjTREC) with standard error ±7.35 years. Within the forensic context, sjTREC' levels can be used to estimate the Egyptian individual's age accurately.     

Investigating Investigators: Examining the Impact of Eyewitness Identification Evidence on Student-Investigators

This research examined the impact of eyewitness identification decisions on student-investigators. Undergraduates played the role of police investigators and interviewed student-witnesses who had been shown either a good or poor view of the perpetrator in a videotaped crime. Based on information obtained from the witness, student-investigators then chose a suspect from a database containing information about potential suspects and rated the probability that their suspect was the culprit. Investigators then administered a photo lineup to witnesses, and re-rated the probability that their suspect was guilty. Student-investigators were highly influenced by eyewitness identification decisions, typically overestimating the information gained from the identification decision (except under conditions that led witnesses to be very accurate), and were generally unable to differentiate between accurate and inaccurate witnesses.

Testing a Novel 3D Printed Radiographic Imaging Device for Use in Forensic Odontology

There are specific challenges related to forensic dental radiology and difficulties in aligning X‐ray equipment to teeth of interest. Researchers used 3D printing to create a new device, the combined holding and aiming device (CHAD), to address the positioning limitations of current dental X‐ray devices. Participants (N = 24) used the CHAD, soft dental wax, and a modified external aiming device (MEAD) to determine device preference, radiographer's efficiency, and technique errors. Each participant exposed six X‐rays per device for a total of 432 X‐rays scored. A significant difference was found at the 0.05 level between the three devices (p = 0.0015), with the MEAD having the least amount of total errors and soft dental wax taking the least amount of time. Total errors were highest when participants used soft dental wax—both the MEAD and the CHAD performed best overall. Further research in forensic dental radiology and use of holding devices is needed.     

Friction Marks of Stabbing Tools in Tires

When receiving a stabbed tire for examination, forensic toolmark examiners can determine whether a suspect tool was used in a specific crime based on class-characteristics and individual-characteristics marks that have been left by the tool on the tire. This study discusses friction marks and their forensic value during the examination of a punctured tire. The term friction mark refers to the noticeable mark around the penetration area on a tire's surface. Tires designed to create high friction when contacting a road. Due to this design, friction is created between the stabbing tool shank and the sides of the hole. As a result of this friction, the shank of the stabbing tool wears the outer layer of tire around the hole. This leaves a friction mark whose general shape reflects the cross-sectional shape of the stabbing tool’s shank. This phenomenon was observed and named by Locke (7) in his evaluation of tire puncture marks with knives. This article demonstrates the same phenomenon with other types of stabbing tools. Test stabs were produced with different tools representing a variety of cross-sectional shapes of shanks, and the resulting friction marks were photo-documented and discussed. Correlations between the various cross-sectional shapes and their corresponding friction marks are shown. Based on friction mark examination, the examiner: (i) can infer suspect tool shank cross-sectional shape with the evaluation of the friction mark shape and (ii) can deduce the maximum dimensions of the shank. This examination simplifies and accelerates the forensic comparison procedure and the investigation time.     

微测序技术检测12个Y—SNP及其遗传多态性

目的应用SNaPshotKit对Y染色体上12个SNP位点进行快速而准确的检测,对四川地区78个汉族男性无关个体进行群体遗传学研究,并对陈旧骨骼和性犯罪案件的相关物证进行检验。方法对SRY2627、SRY1532、M13、M20、SRY8299、Tat、M69及M9、92R7、M17、M19、M112两组共12个Y-SNP位点进行复合扩增,PCR产物经纯化处理后,采用SNaPshotKit试剂结合毛细管电泳技术对单核苷酸多态性进行检测。结果建立了12个Y-SNP位点的微测序快速检测系统,在四川地区人群中发现M9、SRY8299二个位点存在变异。结论复合扩增结合微测序技术能够同时对多个Y-SNP的多态性进行快速而准确的检测,建立的检测系统在法医学个体识别中具有应用价值。     

Development of a Tetraplex PCR Assay for CYP2D6 Genotyping in Degraded DNA Samples

CYP2D6 polymorphism analysis is gaining increasing interest in forensic pharmacogenetics. Nevertheless, DNA recovered from forensic samples could be of poor quality and not suitable for long polymerase chain reaction required to type CYP2D6 gene prior to SNaPshot minisequencing analysis performed to define alleles with different enzymatic activity. We developed and validated following the guidelines of the Scientific Working Group on DNA Analysis Methods a tetraplex PCR yielding four amplicons of 597, 803, 1142, and 1659 bp encompassing the entire CYP2D6 gene to analyze eleven SNP positions by SNaPshot minisequencing. Concordance, sensitivity, and specificity were assessed. The method, applied to thirty‐two forensic samples failed to amplify with long PCR, allowed the amplification of CYP2D6 gene in 62.5% of degraded samples. The new tetraplex PCR appears a suitable method for CYP2D6 analysis in forensic pharmacogenetics.     

Tooth hop variability in human and nonhuman bone: Effect on the estimation of saw blade TPI

Forensic research has demonstrated that tooth hop (TH) is a valuable measurement from saw-cut bones as it can be used to estimate teeth-per-inch (TPI) of a saw used in postmortem dismemberment cases. However, error rates for TPI estimation are still under development and knowledge of how bone tissue affects TH measurements remains unclear. The purpose of this research was to investigate the effects of tissue variability through the use of different taxa on the accuracy and precision of TH measurements in the bone to estimate TPI of the blade. A total of 1766 TH measurements were analyzed from human, pig, and deer long bones cut by two 7 TPI saw blades of different tooth type. Fifty distance-between-teeth measurements before and after sawing were collected directly from each blade for comparison to bone-measured TH to assess potential effects of tooth wear on TH variability. ANOVA and F tests were used to compare mean TH and variance, respectively, by saw-species (i.e., crosscut-deer, rip-deer) and species groups (i.e., all deer, all pig), with significance determined at the p < 0.05 level. TH measurements were converted to usable TPI ranges, which would typically be presented in a forensic report. It is concluded that significant differences in TH (mm) do not necessarily reflect significant differences in associated TPI ranges of suspect blades. Forensic reports should report mean TPI ± 1.5–2.5 TPI while providing a sample size indicating number of TH measured rather than just number of cuts or cut surfaces examined.     

Molecular Identification of Three Indian Snake Species Using Simple PCR–RFLP Method*

Abstract: Three endangered Indian snake species, Python molurus, Naja naja, and Xenochrophis piscator are known to be significantly involved in illegal trade. Effective authentication of species is required to curb this illegal trade. In the absence of morphological features, molecular identification techniques hold promise to address the issue of species identification. We present an effective PCR–restriction fragment length polymorphism method for easy identification of the three endangered snake species, Python molurus, Naja naja, and Xenochrophis piscator. A 431‐bp amplicon from cytochrome b gene was amplified using novel snake‐specific primers following restriction digestion with enzymes Mbo II and Fok I. The species‐specific reference fragment patterns were obtained for the target species, which enabled successful identification of even highly degraded shed skin sample confirming the utility of the technique in case of poor‐quality DNA. The assay could be effectively used for forensic authentication of three Indian snake species and would help strengthen conservation efforts.     

Regularities in Eyewitness Identification

What do eyewitness identification experiments typically show? We address this question through a meta-analysis of 94 comparisons between target-present and target-absent lineups. The analyses showed that: (a) correct identifications and correct-nonidentifications were uncorrelated, (b) suspect identifications were more diagnostic with respect to the suspect’s guilt or innocence than any other response, (c) nonidentifications were diagnostic of the suspect’s innocence, (d) the diagnosticity of foil identifications depended on lineup composition, and (e) don’t know responses were nondiagnostic with respect to guilt or innocence. Results of diagnosticity analyses for simultaneous and sequential lineups varied for full-sample versus direct-comparison analyses. Diagnosticity patterns also varied as a function of lineup composition. Theoretical, forensic, and legal implications are discussed.     

同时鉴定9种肉源种属的复合检测体系的建立及其应用

目的建立一种可以同时鉴定猪、牛、羊、鸡、鸭、猫、狗、鼠和鲤鱼的多重PCR检测方法,并测试其技术性能指标,评估其在法医学或食品安全事件中的应用价值。方法根据上述9种动物的线粒体细胞色素b基因,利用其片段中种间特异性强的序列设计引物,采用毛细管电泳检测平台对各种属PCR扩增产物进行检测,依据扩增片段长度的差异与标记的荧光对初始模板的肉源种属进行鉴别;并通过扩增特异性、实际样本测试、混合样本检测灵敏度等指标评价该方法在实际法医学或食品安全案件中的应用价值。结果经过验证,建立的同时鉴定9种肉源种属的复合检测体系对其中任意一个种属的检测特异性均较高,且灵敏度较强,能够满足绝大多数法医学或食品安全案件中的检测要求。结论本研究建立的肉源种属复合检测体系可以在法医学未知种属样本的鉴定及食品安全肉类掺假等案件中提供帮助。