Rapid and simple sex determination method from dental pulp by loop-mediated isothermal amplification

Sex determination from dental pulp DNA was examined by loop-mediated isothermal amplification (LAMP) method. Amelogenin locus was analyzed for sex determination. A set of four specially designed primers was prepared based on database from Gene Bank, and loop primers were designed to shorten the analysis time. Analysis was performed using 32 dental pulp DNA samples removal from permanent teeth stored at room temperature for 1–25 years after extraction. The X allele was detected in approximately 32 min with real-time turbidimeter and the Y allele was detected in approximately 34 min. Analysis time was reduced to half when using loop primers. Visual detection was also possible as the amplified product showed white turbidity. Sex determination by LAMP method was rapid and simple, and it should prove useful in unknown bodies of mass disasters.     

Skeletal identification by radiographic comparison: blind tests of a morphoscopic method using antemortem chest radiographs

This study investigated the value of antemortem (AM) and postmortem (PM) radiographs of the claviculae and C3-T4 vertebrae to identify skeletons of missing U.S. soldiers from past military operations. In total, 12 field-recovered skeletons and AM chest radiographs of 1460 individuals were used. For each skeleton, examiners analyzed an array of AM chest radiographs (up to 1000 individuals) and attempted to identify the correct PM/AM radiographic match. When examiners were able to compare all images within a single test, only true-positive identifications were made. When AM radiographs were presented one-at-a-time, in sequential order, and without examiners having knowledge of array size, erroneous identifications resulted but they were almost exclusively made by untrained examiners (accuracy = 35% vs. 90% for trained examiners). This study demonstrates the value of chest radiographs for the identification of disarticulated and even eroded skeletons, but only when methods are wielded by trained examiners.     

A novel method for the identification of saliva by detecting oral streptococci using PCR

We have used DNA amplification methods to detect common oral bacterial strains to test for the presence of saliva in forensic samples. Streptococcus salivarius and Streptococcus mutans were detected in various forms of saliva samples, whereas these streptococci were not detected in semen, urine, vaginal fluid, or on skin surfaces. Therefore, we demonstrated that these streptococci are promising new marker for the forensic identification of saliva. Our data indicated that S. salivarius is more reliable than S. mutans as an indicator of saliva presence, because the detection rates for S. salivarius and S. mutans by this method were 100% and 90%, respectively. Furthermore, S. salivarius was detected in all saliva stain samples, whereas S. mutans was only identified in 60% of the stains. Finally, using this method we were able to successfully detect S. salivarius and S. mutans in mock forensic samples. We therefore suggested that this method is useful for the identification of saliva in forensic science.     

Application of superimposition-based personal identification using skull computed tomography images

Superimposition has been applied to skulls of unidentified skeletonized corpses as a personal identification method. The current method involves layering of a skull and a facial image of a suspected person and thus requires a real skeletonized skull. In this study, we scanned skulls of skeletonized corpses by computed tomography (CT), reconstructed three-dimensional (3D) images of skulls from the CT images, and superimposed the 3D images with facial images of the corresponding persons taken in their lives. Superimposition using 3D-reconstructed skull images demonstrated, as did superimposition using real skulls, an adequate degree of morphological consistency between the 3D-reconstructed skulls and persons in the facial images. Three-dimensional skull images reconstructed from CT images can be saved as data files and the use of these images in superimposition is effective for personal identification of unidentified bodies.     

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 new method of reproduction of fingerprints from corpses in a bad state of preservation using latex

In view of the problems arising while fingerprinting corpses in a bad state of preservation, especially in case of mummification and carbonization, the authors propose an innovative technique which uses latex film. To better illustrate the potential of the method, two cases where the latex technique was applied successfully are reported: the first one is a mummified body discovered in a shack on the outskirts of Milan and the second one is the case of a burnt corpse found in a car boot. Such a technique is versatile, easy to apply, and allows the operator to work quickly on cadavers without amputating parts, except in rare cases (i.e., burnt bodies with muscle retraction). By the latex technique, a perfect and enduring negative copy of the fingerprint is obtained, ready to be inked and photographed. The numerous copies produced this way can be inked several times allowing for the repeatability of the procedure and this is crucial in cases of problematic legal identification of corpses. In both the cases illustrated, the fingerprints obtained by the latex technique were useful for identification. The quality was good enough for the automatic fingerprint identification system research system to be applied.     

Rapid and simple GC-MS method for determination of psychotropic phenylalkylamine derivatives in nails using micro-pulverized extraction

A rapid and simple gas chromatography-mass spectrometry (GC-MS) method was developed and validated for the simultaneous detection and quantification of five psychotropic phenylalkylamines (amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, and norketamine) in toenails. After external decontamination, nail clippings were mechanically pulverized with a bead mill and then incubated in methanol under ultrasonication at 50°C for 1 h. The resulting solutions were evaporated to dryness, derivatized, and analyzed by GC-MS. The intra- and inter-day precisions were within 10.7% and 13.9%, respectively. The intra- and inter-day accuracies were -4.2% to 5.0% and -2.4% to 8.4%, respectively. Limits of detection and quantification for each analyte were lower than 0.024 and 0.08 ng/mg, respectively. The recoveries were in the range of 80.6-87.5%. The results indicated that the proposed method is a simple, rapid, accurate, and precise for quantification of five phenylalkylamines in nails. The method was successfully applied to the simultaneous detection and quantification of phenylalkylamines in nail samples of possible drug abusers.     

Development of a fast, simple profiling method for sample screening using high resolution melting (HRM) of STRs

A screening assay has been developed to provide preliminary individualization of crime scene samples thus eliminating expensive, time-consuming short tandem repeat (STR) profiling of nonprobative samples. High resolution melting performed in a real-time PCR instrument is used to detect the slight melting differences between the length and sequence variations of 22 forensic STRs. Three STRs (vWA, D18S51, THO1) were chosen to develop an assay which was optimized for Mg++ concentration, annealing/extension time/temperature, assay volume, and bovine serum albumin addition. The assay was tested for reproducibility, uniformity for genotype, melting profile consistency, effects of inhibitors, and mixture effects. The assay could be used to determine DNA concentration when a standard curve is run simultaneously. Calculations of costs show that the assay can save significant time and money for a crime with many samples or suspects.     

Determining the Source of Equine Bloodstains by Dinucleotide Repeats*

Abstract: A novel multiplex of independent dinucleotide tandem repeat (DTR) loci was previously described that is capable of not only discriminating human and equine DNA, but of identifying a single equine source. We report a case in which a bloodstained syringe and two needles were found during inspection of a barn by inspectors of the Pennsylvania Racing Commissions. Using the multiplex and single‐locus detection, all 21 equine DTR markers were detected in a suspect horse and two evidence samples, indicating the evidence samples came from the suspect animal. Only six markers were detected in the third evidence sample because the volume of blood was limited. Following whole‐genome amplification and single‐locus PCR, the third evidence sample detected a total of 17 markers and the likelihood of identity (probability from suspect horse/probability from a random pacer) was 7.0 × 10⁶. The DTR multiplex has some technical limitations, but it is already practical for casework.     

用等位基因特异扩增技术检测人类补体第八成份C8A DNA多态性的研究

建立补体第八成份蛋白质多态性的DNA检测方法。根据导致C8A多态性的DNA点突变核苷酸差异 ,建立一个检测C8A基因型的特异性扩增方法。采用该法 ,对 10 0份血样本进行检测 ,并同时用传统的检测C8A蛋白质多态性的SDS 凝胶电泳方法进行对照检测 ,结果显示新建立的C8A多态性PCR分型方法不仅快速、灵敏、稳定 ,而且分型结果清晰 ,容易判读 ,适用于法医学中多种检材的C8A多态性分型     

A Large‐Sample Test of a Semi‐Automated Clavicle Search Engine to Assist Skeletal Identification by Radiograph Comparison

In 2014, a morphometric capability to search chest radiograph databases by quantified clavicle shape was published to assist skeletal identification. Here, we extend the validation tests conducted by increasing the search universe 18‐fold, from 409 to 7361 individuals to determine whether there is any associated decrease in performance under these more challenging circumstances. The number of trials and analysts were also increased, respectively, from 17 to 30 skeletons, and two to four examiners. Elliptical Fourier analysis was conducted on clavicles from each skeleton by each analyst (shadowgrams trimmed from scratch in every instance) and compared to the search universe. Correctly matching individuals were found in shortlists of 10% of the sample 70% of the time. This rate is similar to, although slightly lower than, rates previously found for much smaller samples (80%). Accuracy and reliability are thereby maintained, even when the comparison system is challenged by much larger search universes.     

Triton X-100快速提取甲醛固定、石蜡包埋组织内DNA的方法

目的建立一种快速提取甲醛固定、石蜡包埋组织内DNA的方法。方法利用TritonX-100一步提取甲醛固定、石蜡包埋组织内DNA,并具体研究了不同浓度TritonX-100对提取后DNA-STR分型效果的影响。结果成功地一步提取出甲醛固定、石蜡包埋组织内DNA,浓度为1%的TritonX-100提取效果较好。结论利用TritonX-100提取甲醛固定、石蜡包埋组织内的DNA,为快速提取DNA提供了有效的途径,是法科学领域中一种值得推荐的方法。     

A quantitative method for simultaneous determination of 5-methoxy-N,N-diisopropyltryptamine and its metabolites in urine using liquid chromatography-electrospray ionization-tandem mass spectrometry

5-Methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) is a designer hallucinogen derived from tryptamine and is reportedly abused and involved in criminal activities. For the detection of 5-MeO-DIPT use, a liquid chromatography-tandem mass spectrometric method for 5-MeO-DIPT and its metabolites, 5-hydroxy-N,N-diisopropyltryptamine (5-OH-DIPT) and 5-methoxy-N,N-isopropyltryptamine (5-MeO-IPT) was developed and validated in rat urine. The urine samples were pretreated by protein precipitation with acetonitrile and introduced into a BDS HYPERSIL C(18) column (50 × 2.0 mm, 5 μm) for chromatographic separation. Mobile phases consisted of methanol, water, and 1% formic acid, and gradient elution was used at a flow rate of 0.2 mL/min. For the MS detection, multiple-reaction monitoring analysis was adopted. The linear range was 0.01-10 μg/mL, and the lower limit of quantification was 10 ng/mL for all analytes. The intra- and interday accuracies and precisions met the criteria (<15%). The developed method was successfully applied to the drug-treated rat urine.