Japanese Journal of Forensic Science and Technology

Online ISSN: 1881-4689
Print ISSN: 1880-1323
Design of three regions of the 16s rRNA gene. Light blue color boxes represent constant region and dark blue color boxes represent variable region. V1 V9 indicates the variable region number. Two-way arrows represent three regions used in this study, region L: long-range, region M: middle-range and region S: shortrange. One-way arrows represent modiˆed universal primers with tag, see Table 2 for sequences of each primer.
に,模式図を Fig. 1 に示す. . 定量 DNA 溶液中のミトコンドリア DNA の定量は,
BLAST search results of mammalian sample sequences, including reference sequences registered in the RefSeq representative genomes data- base.
BLAST results of mammalian sample sequences, including reference sequences registered in the Nucleotide collection database.
Biological evidence is often found at crime scenes that are from human and various unknown organisms. In some cases, species identification is important for an investigation. Therefore, a direct sequencing method that targets three regions of the 16s rRNA gene was examined to classify 14 mammalian samples. For each mammalian DNA sample, PCR products were analyzed by gel electrophoresis with modified universal primers. Direct sequencing was performed using a BigDye Terminator v1.1 Cycle Sequencing kit and an ABI 3130xl Genetic Analyzer. Each sequence was evaluated by a nucleotide BLAST homology search. The top hits for sequence homology for each sample matched the actual species, or closely-related species. In conclusion, this effective method may be used in routine forensic practices to identify the species of unknown mammalian biological samples, such as from blood, body fluid, tissue, hair, and bone.
Chemical structures of fentanyl and its analogues.  
TIC (a) and EI mass spectra (b) of the trimethylsilyl (TMS) derivatives of b hydroxyfentanyl and b hydroxyl trans 3 methylfentanyl by GC/MS. 1, b hydroxyfentanyl TMS; 2, b hydroxyl trans 3 methylfentanyl TMS.  
Electron ionization (EI) mass spectra of fentanyl and related compounds. 1, fentanyl; 2, acetylfentanyl; 3, thiofentanyl; 4, p ‰uorofentanyl; 5, acetyl p ‰uorofentanyl; 6, a methylfentanyl; 7, acetyl a methylfentanyl; 8, a methylthiofentanyl; 9, cis 3 methylfentanyl ; 10, trans 3 methylfentanyl; 11, acetyl trans 3  methylfentanyl; 12, trans 3  methylthiofentanyl; 13, b hydroxyfentanyl; 14, b hydroxy trans 3 methylfentanyl; 15, remifentanil; 16, sufentanil; 17, carfentanil; 18, alfentanil.  
Simultaneous analytical methods for 18 compounds of fentanyl and its analogues by thin-layer chromatography (TLC), gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS) were developed. In TLC, fentanyl analogues were well separated by using toluene-acetone-28% aq. ammonia (20:10:0.3, by vol.) as a developing solvent. In GC/MS, fentanyl analogues, except for fentanyl and acetyl-α-methylfentanyl, could be separated on the extracted ion chromatograms (EIC) of the characteristic fragment ions of each compound. In LC/MS, fentanyl analogues could be separated on the EICs of the protonated molecule of each compound. All of the fentanyl analogues tested were identified correctly by using the combination of TLC, GC/MS and LC/MS.
From 2012 to 2014 in Japan, a total of 214 cases of motor vehicle collisions were attributed to the use of illegal drugs by drivers. In 93 out of 96 investigated cases, the causative agents were 22 kinds of synthetic cannabinoids (SCs). Those SCs can be grouped into three groups according to the timeline of use and their chemical structures. The first generation SC naphtoyl indole derivatives, such as MAM-2201, were used in 2012 and disappeared by governmental overall regulations in Spring, 2013. Instead, quinolinyl ester indoles (second generation SC, such as 5F-PB-22) and indazole carboxamides (third generation SC, such as 5F-AB-PINACA) appeared thereafter with much stronger potencies. An outbreak of SC occurred in Summer, 2014 with one of the strongest SCs, 5F-ADB. The common signs observed in the SC-abused drivers are impaired consciousness, anterograde amnesia, catalepsy with muscle rigidity, tachycardia, and vomiting or drooling. Since the third generation SCs are extremely potent CB1 agonists (only a small amount is required) and instable in blood, it is very difficult to detect SCs in biological samples. Actually, only in one third of the cases, SC could be detected in blood or urine.
New features for forensic STR typing have become available on 3500 Series Data Collection Software version 4 (DC v4). To investigate impacts of the new features on the interpretation of electropherograms is important to maintain and improve the objectivity and reliability of forensic STR typing. In this study, we assessed and investigated fundamental performance, signal optimization, and pull-up reduction with enabling and disabling the new data optimization features. In fundamental performance verification, although the data generated with the run module-dependent signal optimization showed a tendency to decrease the peak height slightly, all the data set generated from a different combination of enabled and disabled features tested in this study were comparable. In electropherograms generated with the run module-dependent signal optimization, some artifact peaks were observed at lower molecular weight region from the presumed parent allele peaks in the same dye-channel. The number of pull-up peak observations in electropherograms generated with the pull-up reduction feature was decreased compared to when the feature was disabled. However, distributions of distances of a pull-up peak from the corresponding parent peak and morphologies of pull-up peaks were different between the dataset obtained from the pull-up reduction feature enabled and that obtained from the feature disabled in certain pull-up patterns. Therefore, forensic biologists must be aware of those differences when interpreting electropherograms. We conclude that it is useful to use DC v4 with the run module-dependent signal optimization feature disabled and the pull-up reduction feature to obtain electropherograms with less artifact peaks.
The 3500xL Genetic Analyzer will be used for short tandem repeat (STR) analysis because 3130xl Genetic Analyzer has been discontinued. A validation study was performed for the 3500xL to carry out STR analysis of reference samples with Identifiler® Kit. Sensitivity test was performed with 0.125 ng, 0.5 ng, 1 ng and 3 ng DNA as PCR template. All alleles were detected with 0.5 ng, 1 ng and 3 ng DNA input. Any off scale data and noise over 175 RFU that is threshold value of 3500xL recommended by the manufacturer were not observed from 0.5 ng of input DNA. Thus the optimum input DNA for PCR template was 0.5 ng. We also examined Intra-color peak height ratio and heterozygote peak height ratio at 0.5 ng of template DNA. These results obtained by 3500xL showed similar validations with 310 and 3130xl previously reported. Spectral pull-up was examined by inspections of 240 injections data about each 0.5 ng, 1 ng and 3 ng DNA samples in the sensitivity study with a threshold value of 50 RFU. 1,011, 3,210, and 7,056 pull-up peaks were observed in 0.5 ng, 1 ng, and 3 ng, respectively. It was possible to remove all pull-up peaks using a global cut-off filter of 20%. We compared the profiles generated by the 3500xL and 3130xl using 84 samples with 0.5 ng DNA as PCR template. Both 3500xL and 3130xl detected peaks of all alleles contained in them. However, 3500xL also detected noise peaks and genotyping success rate were 71.4-88.1%. When the 3500xL run data were re-analyzed using the 20% filter, the genotyping results showed 100% concordance. The 20% filter is useful to obtain STR profiles with Identifiler® kit using 3500xL from reference samples because reference samples yield high-quality DNA and are collected from a single individual.
Generally, in the analysis of the accident between the car and the pedestrian, an index called Wrap Around Distance (WAD) is used to quantify to the positional relationship of the pedestrian's head because the pedestrian falls so as to wrap around the front shape of the car. There are a few reports in Japan that the relationship between the WAD ratio and the impact speed was expressed by linear regression based on the assumption that the position where the pedestrianʼs head collides moves to the rear of the car as the impact speed of the car increases. However, the coefficient of determination in the linear regression is so small that the behavior of the pedestrianʼs head during the collision was not properly described. It is inappropriate for the accidental reconstruction to apply WAD in estimating the impact speed of the car. We created the mathematical model that predicted the positional relationship between the pedestrianʼs head and the car analyzing 56 accidents. Our model described in detail the behavior of the pedestrianʼs head by considering both the behavior of the pedestrian during the collision and the deformation of the car. As a result, our model indicated that the behavior of the pedestrianʼs head changed the distance from the height of the front edge of the hood to the pedestrianʼs center of gravity, and the position restricted the upper limit by visualizing the simulation results for 7 data.
Blood acetaldehyde levels are often measured to elucidate individual differences and pharmacokinetics of alcohol metabolism due to the gene polymorphisms of aldehyde dehydrogenase (ALDH2). Blood acetaldehyde levels are of great interest in the field of addictive behaviors, particularly drunk driving behaviors. Conventional blood acetaldehyde concentration measurements are direct and derivatization analyses. Because direct analysis is problematic in that acetaldehyde disappears during storage due to its high volatility and reactivity, derivatization analysis is often performed for acetaldehyde determination in biological samples. The conventional derivatizing method using 2,4-dinitrophenylhydrazine for determination of aldehydes unfortunately produces interfering substances from ketones and carboxylic acids. Therefore, we applied the derivatization method using 9,10-phenanthrenequinone (PQ), reported recently for the interference-free determination of aldehydes. As a result of optimizing the derivatization conditions, a linear calibration relationship (R²=0.9994) in the range of 3-100 μM indicated strong results. The limit of detection and quantification was 1 μM and 3 μM, respectively. The intra- and inter-day precision were both below 13.0% and accuracy was in the range of -8.5% to 2.5%. The extraction recoveries of acetaldehyde in human plasma ranged from 80.4% to 106.8%. The derivative was stable at 4 ℃ for 2 days. When acetaldehyde was added in human plasma and derivatized with PQ, acetaldehyde could be quantified by liquid chromatography-mass spectrometry. However, after drinking alcohol, acetaldehyde was measured in healthy subjects with ALDH2*1/*2, but not with ALDH2*1/*1. Acetaldehyde was difficult to detect in subjects with ALDH2*1/*1, as they are rapid metabolizers of acetaldehyde resulting in low concentrations of acetaldehyde. Acetaldehyde was detected in those subjects with ALDH2*1/*2, however, the measured acetaldehyde levels varied between the analytical methods.
Analytical results for trace elements in biological sample can provide useful information to prove that a victim had taken toxic inorganic elements. In this study, we aimed to establish a detection method to detect toxic elements in a blood stain. Simulated blood samples prepared by mixing thallium standard solution in horse hemolysis blood were dropped and dried on a filter paper. Micro X-ray fluorescence spectrometry and scanning electron microscopy/energy dispersive X-ray analysis could detect only major elements in the blood stain but not the added thallium. Laser ablation-inductively coupled plasma mass spectrometry made it possible to detect those elements in the blood stain. Calibration curve obtained by measuring filter paper made from aqueous solution or horse hemolysis blood containing thallium exhibited good linear relationship in the range of 0~500 ng/mL. The present method can be used for the qualitative screening of toxic elements in blood stains.
Since we are always surrounded by ceramic products, it may be collected from the scene of the crime. In this study, we aimed to establish the discrimination methods for these samples. From the elemental mapping obtained by X-ray fluorescence analysis, Ca and Zn were recognized at the surface, Si, K, Ti and Fe were found inside. However, since scarce information was provided it was difficult to find a difference among samples by X-ray fluorescence analysis. We performed trace elements analysis using laser ablation-inductively coupled plasma mass spectrometer (LA-ICP-MS) for ceramic samples. Some elements such as Sc, Ti, V, Mn, Fe, Co, Zn, Ga, Rb, Sr, Zr, Cs, Ba, La, Ce, Pb, Bi were found from the inside of the ceramics. Among them, V, Co, Ga, Rb, Sr, Cs, Ba and Pb were selected to calculate index and didn't have mass spectral interference with other elements and uneven distribution. 23 kinds of commercially available samples were collected and analyzed by LA-ICP-MS. As the tendency that the signal intensity ratio differed among different samples was observed, forensic discrimination could be performed using this intensity ratio as an indicator. Discriminations between samples were performed by the threshold based on standard deviation called 2SD method. Moreover discrimination results were evaluated and the number of discriminated pairs among all were combinations. Only about 32-84% was discriminated when using single index, but they were improved to about 95% by the combination of multiple indices. When taking account the origin of the samples, 227 of 228 pairs (99.6%) which clearly originated differently were discriminated. This method shown in this paper is quite effective for the discrimination of ceramics.
In forensic investigations, ABO genotyping via detection of single nucleotide polymorphisms (SNPs) in the ABO gene is often performed to estimate the ABO phenotype of case samples. This study involved the developmental validation of the ABO genotyping method performed by multiplex real-time PCR using TaqMan probes targeting major O or B allele-specific SNPs. We first evaluated the genotyping accuracy of this method by using 2 ng of blood DNA and confirmed that the ABO genotypes could be correctly determined from all samples. To validate the effect of unbalanced amplification of heterozygous samples on the genotyping accuracy, we analyzed various amounts (0.03125-1.0 ng) of two blood DNA samples of the BO genotype, which were heterozygous for both O and B allele-specific SNPs. No false genotypes were observed, even in the analysis of low-template DNA, although 1 ng of template DNA was required for stable detection. We also analyzed BO blood DNA mixed with humic acid or hematin. Although inhibition of PCR by using high concentrations of both inhibitors caused “no amplification” results, no false genotypes were observed under any condition. We confirmed that the correct genotypes could be obtained by using 1 ng of DNA from blood stains stored at room temperature for 2-36 years. Finally, we analyzed the DNA from five animal species (chimpanzee, Japanese macaque, dog, cat, rat) and confirmed that several probes reacted strongly to the primate DNA and weakly to the feline DNA, indicating that careful attention should be paid in the analysis of samples that may be derived from these species. Because the genotypes of multiple samples can be simultaneously determined within about 30 min after DNA quantification, this method is expected to be useful for forensic ABO genotyping.
Threshold range of each genotype group and genotypes resulting from a combination of determined genotype groups. 
ABO genotyping of aged stains and bone samples using the DNA chip method. 
DNA chip analysis with various amounts of BO genotype DNA.
genotyping of aged stains and bone samples using the DNA chip method.
In forensic investigations, ABO genotyping is often performed to obtain information for individual identification. This information is especially effective in the analysis of highly decomposed samples with which the serological method for ABO typing cannot be used due to ABH antigen deterioration. The authors previously reported on the development and improvement of a microarray-based method that can be applied for simultaneous ABO genotyping and human identification using two pairs of probes to detect major O or B allele-specific single nucleotide polymorphisms (SNPs) in the ABO gene and a probe to determine the human-specific D17Z1 sequence. In this study, a probe pair for B allele-specific SNPs was redesigned and selected because those used in the previous study appeared less than optimal in regard to specificity between BB and B heterozygous genotypes, which could give rise to false results. When DNA from bodily fluids (blood, saliva, semen and vaginal fluid), tissues and blood from cadavers, and blood stains were analyzed using the redesigned chip, the O and B index values, which reflect fluorescent patterns of each probe pair, could be clearly separated according to genotype. On the basis of these results, threshold values for each index were set and their use for genotype determination in analysis of aged stains and bone samples was verified. Finally, validation was performed with respect to the effect of the amount of input DNA for ABO genotyping by analyzing 0.0625 to 0.5 ng of DNA with the BO genotype. The results demonstrated that more than 0.25 ng of DNA is necessary for accurate genotyping with this method. Due to its simplicity, rapidity and accuracy, the approach is expected to be useful for ABO genotyping in forensic investigations.
Appraisal related to charring of wood may be conducted for arson cases, especially fires with a small scale of burning. When wood is carbonized, a distinctive form is observed, such as the surface turning black or cracking, but no clear appraisal method has been established, and the criteria set by individual appraisers have not been established. The current situation is that appraisal is conducted based on these individual appraisal methods. However, it is expected that the presence or absence of carbonization of the wood can be objectively determined by measuring the infrared absorption spectrum of the burned wood using a Fourier transform infrared spectrophotometer. Therefore, in this study, an experimental model was constructed on the assumption of an actual arson case, a combustion experiment was performed on wood, which is widely used as a building material, and data on the obtained carbides were examined and objectively carbonized. We have devised a carbonization appraisal method. In this study, in addition to FT-IR analysis, SEM-EDX analysis and XRD analysis were also used to analyze detailed carbides. As a result, FT-IR analysis showed that objective wood can be identified for carbonization. However, when heated by a high temperature flame, the wood transitions to complete carbide, but when heated by a relatively low temperature flame as in the experimental model implemented in this study, it is difficult to transition to complete carbide.
When traffic accidents happen, investigators observe the state of the vehicle at the crash site of the accident. Here the energy conservation law can be used to estimate the vehicle collision speed. Since the collision speed of the vehicle can be estimated from the energy of its deformed volume, investigators observe and measure the vehicle's crash state. However, when a vehicle collides into traffic signs, both the vehicle and sign will be deformed. Consequently, to estimate correctly the vehicle collision speed, the energy absorption capacity of the signs should be taken into account with the vehicle deformation after the crash. In this study, to improve the estimation of the collision speed of the vehicle, the drop weight tests were done to examine the absorption energy of the signs. The traffic signs used in this test are a road sign, a road mirror and a guard-rail on the roadside. From test results, it can be concluded that the input energy is linearly proportion to the deformed angle of the sign. Also from this test, the relationships between energy and deformed angle of the signs can be formulated. Identification for estimating the vehicle collision speed at an accident hereafter can be done properly with ease.
The motorcycle has a high running stability. Therefore, the motorcycle can keep running without the rider before and after a traffic accident. For Traffic accident reconstruction which involves an unmanned motorcycle, we needed the deceleration value of the motorcycle during running without a rider. Running without a rider is in much the same state as running with the engine brake on. Therefore, we investigated performance of motorcycle engine brake to use for analysis of traffic accident. Engine displacement of the motorcycles with manual transmission (MT) we used was from 250 cc to 1200 cc. And that with automatic transmission (AT) was 50 cc and 250 cc. We examined coasting tests in various speeds and various gears. In the result, we found that the motorcycle with MT speed bear a direct proportionate relationship to deceleration of engine brake running. And then, we found that speed change per unit distance doesn't depend on the motorcycle speed in case of high speed running. However, speed change per unit distance decreased in the case of low speed running by the motorcycle with MT. Simultaneously, the motorcycle with AT increased the value of speed change.
Motorization produced traffic accidents and its conflicts to be brought into the court of law. The field of accident reconstruction was born to cope with these litigations. Essentially, traffic accident is a crime of negligence. Punishment and insurance payment depend upon the assessment of the case. Vehicle speed plays an important role in the ratio of fault and plays an important role as a standard of fault authorization. An analysis method has been developed ever since. Now in Japan, traffic accident is punished with a special law independent from the existing penal code. Speed analysis has become a more important factor under this law. Therefore speed analysis has a long historical path and still plays an important field within forensic science. Fundamental method of speed analysis has been long based on the application of basic physics principles. Recent evolution of electrical devices brought another approach to the speed analysis. Images of CCTV, drive recorders, and vehicle mounted electrical data are offering a good chance to analyze vehicle speed. We would like to review the speed analysis of the traditional methods and overview the trend of recent methods of the new era.
抄録 Currently, polygraph examinations in Japan use the concealed information test (CIT) to determine whether a suspect knows specific details of a crime. The present study examined the accuracy of the CIT as a memory detection technique in a mock-theft experiment. Participants were randomly assigned to either an encoding or non-encoding group. An expert polygrapher who was not informed of the group assignments, conducted a CIT that consisted of two questions. One inquired about a card number chosen by the participant, and the other regarded an item that had been stolen. Analyses focused on the second question. Roughly 20% of cases were judged inconclusive while sensitivity and specificity for the remaining cases were 86% and 95%, respectively. Analysis was repeated using modified Lykken scoring, and rates of inconclusive cases, sensitivity, and specificity by this method were 25%, 83%, and 91%, respectively.
Search area and average rank of each group, function and distance measure.
The accuracy of geographic profiling for predicting a serial offender's home/base location was compared by using three different distance measures—the Euclidean distance, the Manhattan distance, and the Shortest route distance—using the data of 1,856 crimes committed by 124 residential burglars in Northern Tohoku area of Japan from 2004 to 2015. Logarithmic and the negative exponential coefficients were estimated as the distance decay function for each distance measure by using leave-one-out cross-validation. Also, search areas were calculated to compare the accuracy of geographic profiling. Results of the Friedman's test indicated significant differences in search areas of the three distance measures for the wide area group which consisted of offenders having a long distance between crime locations. The search area when utilizing the Shortest route distance was the smallest for the logarithmic function, whereas the search areas using the Euclidean distance and the Shortest route distance were smaller than the Manhattan distance for the negative exponential function. Results of the narrow area group did not indicate significant differences in search areas for the three distance measures. Therefore, it was concluded that geographic profiling might be improved by using the Shortest route distance when calculating the probability distribution for offenders committing crimes in a wide area that includes many edges, such as rivers, railroads, and mountains, as well as paths such as bridges and railroad crossings.
We selected a group of men who acknowledged sexual interest in prepubescent girls based on the Sexual Desire Scale for Males (SDS-M). Subjects were members of the general population aged 18 years and above who were not in prison (N=573). The relationship between acknowledging sexual interest in prepubescent girls and factors such as their experience of sexual offence, personality traits, cognition towards women and use of pornography were examined. It was estimated that 10% of survey subjects had acknowledged sexual interest in prepubescent girls. Statistical analysis showed that acknowledging sexual interest in prepubescent girls was significantly related to experience of sexual offence against women, some of sexual desire, some of personality traits and acceptance of sexual violence. A significantly higher proportion of men who acknowledged sexual interest in prepubescent girls had experience of sexual offence against women and of using child pornography compared to those who did not. The results of this study suggest that an understanding of sexual interest in prepubescent girls requires the perspectives of cultural and social learning.
The adhesives of seventeen acrylic pressure sensitive cloth adhesive tapes and seventeen kraft adhesive tapes were analyzed by pyrolysis-gas chromatography/mass spectrometry(Py-GC/MS) at 500°C. The main pyrolysates were butyl acrylate (BA), 2-ethylhexyl acrylate(2EHA), ethyl acrylate(EA), methyl methacrylate(MMA), tolylenediisocyanate(TDI) and the pyrolysates of an aromatic petroleum resin(C9 Resin). The acrylic adhesives were classified into twelve groups according to their pyrolysates.
We examined the decomposition behavior of 1-acyl-substituted derivatives of d-lysergic acid diethylamide (LSD), such as 1-cyclopropanecarbonyl LSD (1cP-LSD), 1-acetyl LSD (ALD-52), and 1-propionyl LSD (1P-LSD) through gas chromatography/mass spectrometry (GC/MS). It was reported previously that in methanol, 1cP-LSD completely decomposed into LSD during GC/MS. We found that in methanol, 1cP-LSD remained undecomposed during GC/MS, and the extent of decomposition varied based on the analyses performed in this study. As a result of a detailed examination, we deduced that the decomposition occurs at the inlet, regardless of the inlet temperature or the type of the inlet liner. We observed that the peak areas of 1cP-LSD decreased with the deterioration of the inlet liner, and this was considered to be a cause for the variation between different analyses. While the acetonitrile solution of 1cP-LSD provided relatively robust results, the other examined solvents showed a significant decomposition of 1cP-LSD and/or a sequent decrease in the peak area of 1cP-LSD with time after the replacement of the inlet liner. ALD-52 or 1P-LSD in acetonitrile were stable during GC/MS, however, they were unstable when methanol was used as a solvent, similar to 1cP-LSD. This suggested that a similar decomposition and/or a sequent decrease in the peaks of ALD-52 and 1P-LSD during GC/MS can be expected.
Crimes using sleeping pills with malice are occuring continuously. Although it is essential to identify the brand name of sleeping pills in criminal investigations, the recent diffusion of generic drugs has led to a problem that the brand name of the sleeping pills used by suspects cannot be narrowed down from the identification of the active ingredients only. To solve this problem, we have tried to develop a method for identifying the manufacturer of a sleeping pill by analyzing the pharmaceutical additives in left-over beverages. Etizolam tablets 0.5 mg which are manufactured by 16 pharmaceutical companies were selected as the objects of study. The powdered Etizolam tablets were mixed into water and beverages and then discriminated whether the pharmaceutical additives are contained or not with six analytical methods, including Pyrolysis-gas chromatography-mass spectrometry. As a result, it became possible to identify almost all of the pharmaceutical companies via a combination of contained pharmaceutical additives. This analysis method to identify the pharmaceutical companies by analyzing the pharmaceutical additives is the first attempt in our extension research. We think that this method can be applied to many other medicines, and expect that this method can contribute much to identifying or narrowing down suspects.
Forensic samples may include small plant fragments collected as trace evidence that are examined by microscopy and DNA analysis. These fragments are often recovered on adhesive tapes or sheets; as such, recovery must be carried out carefully so that important morphological features, including thorns or trichomes, are not destroyed. In this study, we investigated the use of organic solvents for the recovery of small plant fragments from adhesive tapes and sheets. Particularly, we examined the influence of the solvent and of the adhesive compound on the DNA analyses. Therefore, our goal was to determine an appropriate recovery method for small plant fragments that did not have a negative impact on critical forensic analyses. Plant samples, including seeds and leaves, were attached to adhesive sheets or tapes and recovered with solvents such as water and organic solvents. The extent of recovery and the influence of the adhesive compound and solvent on the subsequent DNA analyses were examined. After the immersion of plant samples in the solvent to detach them from the adhesive compound, DNA extraction and polymerase chain reaction (PCR) amplification were performed. Among our findings, we determined that plant samples on most types of adhesive sheets can be recovered with tweezers alone and are appropriate for microscopic evaluation and DNA analysis. Organic solvents were used to recover samples attached to sheets with strong adhesives. This method had no discernible impact on DNA extraction or the subsequent PCR analyses. Thus, we concluded that small plant fragments attached to adhesive sheets or tapes can be recovered for forensic examination with a variety of methods, including the use of organic solvents.
Adulteration of foods and beverages with drugs and poisons has frequently occurred with or without intention in various incidents, and has therefore been one of the most important subjects in forensic science. The purpose of this study was to develop a simple, rapid and accurate screening method for allegedly adulterated foods and beverages using probe electrospray ionization mass spectrometry (PESI-MS) with a simple pretreatment procedure. Seven dishwashing detergents, four pesticides and four sleeping pills were selected as foreign substances, which were added to four kinds of beverages: barley tea, sports drink, carbonated beverage and milk tea. These simulation samples were subjected to PESI-MS to investigate the optimal analytical conditions for effective detections of foreign substances. A two-fold dilution of the samples with 2-propanol was adopted as pretreatment for PESI-MS to achieve higher sensitivity. As a result, surfactants, which are the main ingredients in dishwashing detergents, were detected with high sensitivity. The main ingredients were undetectable for some of the pesticides and sleeping pills analyzed; however, coexisting additives like surfactants or lactose were detected with high sensitivity indicating that the tested beverage had been adulterated by a foreign substance. Background subtraction of the reference beverage mass spectra from those of the samples also enabled clearer and more reliable determination of the adulterants. The presented PESI-MS method enables accurate screening in two minutes per sample with only a very simple pretreatment, applicable for large-scale screening of adulterants in foods and beverages. This method can serve as an extremely effective screening tool in combination with confirmatory instrumental analysis methods for identifying foreign products.
Offline writer verification is an important security measure and is related to human traits. However, even with modern techniques, maximizing the amount of information from the limited number of available samples remains a challenging task. We propose a new offline writer verification system inspired by the expertise of forensic document examiners. The system combines the shape and pen pressure information in multiple characters captured by a multiband image scanner. From the visible images, the shape information is extracted as a weighted direction code histogram, and the local pen pressure information (which is based on ink intensity) is extracted as local directional pattern features with adaptive zoning. From the infrared images, the global pen pressure information (which is based on the writing indentations) is extracted as first- and second-order texture statistics. In an experimental comparison study at less than 0.1% error rate, the proposed system reduced the number of required Japanese Kanji characters from six (in the conventional system) to three. After combining these techniques, the error rate per character (averaged from 54 volunteers) was reduced from 4.8% in the conventional method to 1.3%.
Gas chromatographic mass-spectrometric analysis was performed for chemical warfare agents and related compounds including sarin, soman, tabun, VX, mustard gas, lewisite 1, 2-chloroacetophenone, o-chlorobenzilidenemalononitrile and capsaicin, under electron ionization and methane chemical ionization conditions using apolar and polar capillary columns. It was possible to identify the tested compounds with respects to their retention indices and mass spectra. Under the analytical conditions of split ratio (50:1), electron ionization and scan mode data aquisition, the limit of detection ranged from 0.06 to 7 μg/ml, except for the low detection sensitivity of lewisite 1.
An MX908 portable mass spectrometer utilizing low-vacuum (high pressure) mass analyzer was evaluated for the detection of chemical warfare agents. The MX908 mass spectrometer accommodates volatile agents like sarin (GB) in the vapor mode and low-volatile agents like VX in the trace mode. We verified that VX could be detected in the trace mode, however a Nomex swab was superior to the original PTFE swab in terms of the alarm limit of VX. For the screening of VX on stainless-steel or plastic surface, we recommend to wet the Nomex swab with 2-propanol (10 μL) prior to wiping the surface or drop 2-propanol (10 μL) on the surface and wipe with the Nomex swab immediately. The optimized wiping methods could detect 100 ng of VX on both of the surfaces. GB was detected at 0.25-0.50 mg/m³ in the vapor mode, which was compatible with a common ion-mobility spectrometry instrument (LCD 3.3). Tabun (GA) was properly detected at 1 mg/m³, however neither MX908 nor LCD 3.3 could discriminate soman (GD) and cyclohexylsarin (GF) at 1 mg/m³. Sulfur mustard (HD) could not be detected at 64 mg/m³. Because significant loss (18-54%) during vapor preparation presumably due to the adsorption on the surface of the sampling bag and the transfer tube was observed, actual sensitivity would be superior to the above-mentioned nominal values.
We ascertained that one of the lachrymator, allyl isothiocyanate (AITC), reacted with alkyl alcohol to form O-alkyl allylthiocarbamate. AITC was incubated with methanol, ethanol (EtOH) or 2-propanol, respectively, and analyzed by gas chromatography/mass spectrometry (GC/MS). On the total ion chromatogram of the reaction mixture, one peak appeared which was different from AITC, and the molecular weight was 131, 145 or 159, respectively. The reaction product of AITC with EtOH was prepared from the mixture. By 1H nuclear magnetic resonance (NMR) analysis, six pairs of the signals which resembled each other appeared at room temperature and some of the paired signals overlapped by heating. It was suggested that the reaction product of AITC with EtOH was O-ethyl allylthiocarbamate, and that it was composed of the mixture of conformational isomers at room temperature.
Ricin, which is a proteinous toxin contained in castor beans, is one of the chemical warfare agents and strictly regulated by law. Regardless of the high toxicity, they have been used in some cases such as attempted murder cases due to the easy accessibility of castor beans. For these cases, detection of ricin is important for case control and proper remediation or decontamination. In this work, we have investigated the applicability of Bio-Threat Alert (BTA) test strips for the detection of ricin in beverages. By using BTA test strips, standard ricin protein in various beverages as low as 0.1 μg/mL could be detected by visual detection or by Guardian BTA Test Strip Reader. The calibration curves for ricin in tomato juice and cafe au lait were constructed using sample values obtained from Guardian BTA Test Strip Reader, which is the intensity of the color developed by the reaction of ricin and antibody. At high concentrations, the response seemed to be saturated. From the beverages added with mashed castor beans, ricin could also be detected by BTA test strips. Some beverages such as milk showed interfering behavior, but the reason seemed to not only come from the total protein amount but other factors.
D19S433 repeat region and primer binding site in this study. Position`rPosition`r' indicates allele G to A mutation position described in previously study. Primers of seq_fwd and seq_rev are used for direct sequencing. Primers of them and typ_G32A_rev are used for SNaPshot genotyping. Primers and probes of taq_fwd, taq_ rev, probe_G and probe_A are used for TaqMan genotyping. These primer designs are based on Genbank reference sequence (AC008507.11).
Electropherogram of D19S433 and FGA peaks tested with PowerPlex Fusion System, labeled by CXR-ET ‰uorescent dye. Sample 1 3 (a) has normal homozygous wild allele, sample 1  4(b) has normal heterozygous peaks of wild alleles, and sample 1 5(c) (one of their children) has normal heterozygous peaks of wild alleles as well as his mother.
Electropherogram of D19S433 downstream ‰anking region by direct Sequencing. Sample 1 3 (a) has homozygous G32A mutation, sample 1 4(b) has no mutation in this region, and sample 1 5(c) (one of their children) has G32A heterozygous mutation. These electropherograms are aligned with Genbank reference sequence (AC008507.11) by SeqScape v2.5 software.
In the Japanese population, D19S433 silent allele is rarely detected in cases of testing with commercial STR kits. The silent allele causes inconsistency of STR typing results between kits and false negative parentage despite the true biological parentage. The cause of this problematical mismatch is reported that the mutation is a base change (G>A) 32 nucleotides downstream from the 3′ end of the AAGG repeats (G32A), so reverse primer in STR kits fail to anneal to the binding site, consequently no STR peak or extremely low peak is detected. In this study, volunteers originated from 4 silent-allelic pedigrees are examined whether the silent allele was judged by AmpFlSTR Identifiler Plus PCR amplification kit, PowerPlex Fusion system, and GlobalFiler PCR amplification kit, furthermore they carry G32A mutation or not by direct sequencing, SNaPshot genotyping, and TaqMan genotyping. In conclusion, it has been identified that all silent-allelic peaks are caused by G32A mutation and followed by Mendelian genetics. Actually, some factors influence the formation of silent allele, such as primer binding fidelity, improvement of other PCR reagents, and PCR cycle conditions. When the suspected silent-allelic peak appears, additional tests with multiple STR kits which containing alternative primer will be recommended.
Discrimination between allele peaks and stutter peaks is important in STR typing for forensic purposes. In minus stutter, it is known that there are correlations between alleles and stutter ratios, but there are few reports on other types of stutter such as plus stutter. In this study, we examined the relationship of alleles to stutter ratios in Y-STR typing using the YfilerTM Plus PCR Amplification Kit. DNA was extracted from blood samples collected from Japanese males and multiplex PCR amplification was performed with 1 ng DNA template. In minus stutter, high correlation coefficients (r>0.7) between alleles and stutter ratios were observed in 23 of 25 markers. Meanwhile, in other types of stutter, only the forward stutter in DYS392 showed such a high correlation coefficient. These results can be used for interpretation of electropherogram data in forensic Y-STR typing.
Tri-allelic peaks are rarely detected from single-source DNA in the case of testing with commercial STR kits, while homozygous or heterozygous peaks are frequently observed at each locus. Tri-allelic patterns can possibly occur in healthy people, and the peak balance of various tri-alleles from different origins can cause some problems that discern from artifact peaks affecting the result of STR typing, or difficulties in the evaluation of kinship. In this study, different samples from a volunteer with D21S11 tri-alleles (alleles 26, 29, and 30) were tested using routine STR analysis methods. The peak balances of the tri-allele varied significantly between samples, therefore a type 1 tri-allelic pattern caused by somatic mutation in the early stages of differentiation was possible because the sum of the lower two peak heights was roughly equal to the highest peak height in every case. Direct sequence analysis of the individual's family members revealed that the tri-allelic pattern was not inherited from the mother, nor was it passed down to the daughter. In addition, a mutated form of one of the tri-alleles and its mutated repeat unit and numbers were identified. When tri-allelic peaks are suspected, it is essential to analyze not only intra-locus peak balance but also the whole electropherogram profile. This means that STR typing is necessary considering the fact that pull-up peaks or stutter peaks could resemble tri-allelic peaks.
The field test is a powerful tool for prompt identification of drug possession. The Scott test, which is widely known as the field test for cocaine, has a serious problem with specificity. The test results in false-positive for some drugs, and these false-positives may lead to erroneous arrests. In order to solve this problem, the authors developed a new field test for cocaine that is applicable for the criminal investigations of drugs. In the first step, blue precipitates appear in a cobalt thiocyanate solution in a known reaction. Then, in the second step, the blue coloration is faded by an additive and resulting in a colorless solution. For this, 2-Iodosobenzoic acid was the most effective additive. This new procedure combined with the Liebermann reagent or Mandelin reagent makes it possible to clearly discriminate between cocaine and other drugs that have previously resulted in false positives with the Scott test. Even a police officer without drug expertise can use this new test, which only requires adding reagents and stirring.
The influence of liquid cleaners on the inner wall coating of aluminum bottle cans was examined by a combination of 20 cleaners and 21 bottles. The bottle cans were classified into three types by their inner resin coatings. The coatings were an epoxy resin, another type of epoxy resin and a polyester resin. After a small portion of liquid cleaners were dropped on the epoxy resin inner coatings of bottles, one of the cleaners generated hydrogen bubbles but the other 19 cleaners caused no effect. This phenomenon was not observed even after about twenty hours when using other liquid cleaners. A mixture of alkalis (NaOH and KOH) and a solvent (diethylene glycol mono-n-butyl ether) in the components of the liquid cleaner in question brought the same phenomenon, and the concentration of the solvent and the time taken to start bubbling were correlated. Therefore, we concluded that because the solvent denatured the inner resin coating of aluminum bottle cans, the alkalis were made accessible to aluminum and generated hydrogen bubbles as a result.
We experienced a case of successful identification of an unknown body found at breakwater based on root canal treatment. After matching the dental findings of the body to the treatment history of individual's dental records, 23 teeth showed agreement in findings. Although 8 teeth did not agree in findings, they were consistent in terms of dental treatment history. There was inconsistency in the remaining tooth. This tooth was determined as intact, but the dental records indicated the existence of a resin composite restoration on that tooth. However, that inconsistency never became a critical determinant factor. Comparison of periapical radiographs of the body with the dental records revealed that the right mandibular first premolar teeth showed considerable similarity to the images of a broken endodontic instrument and a alveolar bone resorption caused by the leakage of root canal sealer at the middle of the root. Given the above information, we concluded that the identification as the same individual is reasonable. It was thought that a case where the findings of a dental medical accident helped to confirm the identity was unusual.
抄録 To obtain DNA typing results from forensic biological samples is occasionally difficult, since they are complex and diverse. AmpFℓSTR® Identifiler® Plus PCR Amplification Kit (IDPlus) was developed to improve the success rate of DNA typing from such samples by Applied Biosystems. Forensic biologists must be cautious in DNA typing when considering the characteristics of the Kit used for analyzing in such samples. Therefore, to reveal to the characteristics of IDPlus Kit is very important to increase the reliability and probative value of DNA typing in the trial and investigation. In this study, IDPlus is evaluated for the characteristics and the DNA detectability. We also compared IDPlus with AmpFℓSTR® Identifiler® PCR Amplification Kit (ID) by analyzing samples difficult to obtain DNA types. The results from the PCR inhibitor study using bloodstain on soil or denim indicated that IDPlus was more resistant to PCR inhibitors than ID. In the study of analyzing degraded DNA, there were no significant differences to be found except for the sensitivity. The peak corresponding to insufficiently amplified products due to a point mutation on primer binding site was higher using IDPlus than ID in electropherogram. Also the anomalous peaks which were observed in analyzing same mammal animal DNA samples were higher. Hence, IDPlus is considered to be very effective leverage to samples analysis which is difficult to detect allele due to PCR inhibitors. The primer binding ability to the template DNA was altered when using IDPlus compared with the ID so this should be taken into consideration. Forensic biologists must be aware the possibility of the discordance of DNA typing results between IDPlus and ID. From the results of this study, IDPlus was considered to be utilized to analyze forensic samples difficult to detect allele using ID.
A validation study was performed on the new STR (Short tandem repeat) multiplex PCR Kit for human identification: AmpFℓSTR® Identifiler® Plus PCR Amplification Kit (IDPlus) released from Applied Biosystems. IDPlus contains the same fifteen loci as AmpFℓSTR® Identifiler® PCR Amplification Kit (ID) which is currently widely utilized in forensic DNA analysis. Consequently, IDPlus has the same discrimination ability as ID. According to the manufacturer, this kit has higher sensitivity, more resistant to PCR inhibitors and less background noise in electrophoresis. Thus, IDPlus was expected to be applied to forensic samples difficult to DNA profiling using ID. An applicability of IDPlus to forensic STR analysis was evaluated. Our study confirmed that IDPlus was more sensitive than ID. IDPlus has about 1.4 times to 1.5 times higher peak than ID when using the same PCR cycle number (28) for both kits. While ID has only one PCR protocol, IDPlus has two PCR protocols differing in cycle number: 28 and 29. To clarify the basic ability we compared heterozygous peak height ratio, stutter, intra-color balance, inter-locus balance among IDPlus to both protocols and ID from 94 individual DNA samples. Lower peak height ratio and the larger standard deviation in heterozygous samples were observed when using IDPlus 29-cycle compared to IDPlus 28-cycle. There was no significant difference between ID and IDPlus 28-cycle and between ID and IDPlus 29-cycle about the peak height ratio. Stutter ratios significantly were different in some loci between ID and IDPlus. Although Applied Biosystems supplies one stutter ratio to filter peaks for each locus to IDPlus in spite of having two PCR protocols, it was confirmed that the same stutter ratio could proper filter out stutter peaks for both cycle numbers. With regard to intra-color balance and inter-locus balance, IDPlus 28-cycle tended to be the highest in balance and ID tended to be the lowest in balance across all the samples and colors. Our validation study indicates IDPlus kit is useful in forensic applications, especially for analysis of trace DNA samples when using 29 cycles. However, forensic biologists must be cautious in interpreting DNA profiles obtained using IDPlus 29 cycles, because peak balance in heterozygous samples tended to be imbalanced when using 29 cycles compared to 28 and ID.
In-line solid phase extraction capillary electrophoresis-mass spectrometry (In-line SPE-CE/MS) has been developed for the screening of methamphetamine (MA) and amphetamine (AP) in hair. A Teflon tube packed with solid phase extractant was connected In-line to the capillary. The detection sensitivity of this method was about 100 times higher than that of common stacking methods. Since the filtrate of digested hair samples could be injected directly into the In-line SPE-CE/MS system, enzyme-based digestion techniques were investigated for the pre-treatment of hair samples. As a result, we found that hair was mostly digested with proteinase K, along with dithiothreitol, urea and TNE buffer (pH 8.0, 10 mM Tris, 100 mM NaCl, 1 mM EDTA) at 55℃ in an hour. Finally, hair samples spiked with MA and AP were analyzed by the developed In-line SPE-CE/MS method. The limits of quantification were 0.08 ng/sample for MA and 0.18 ng/sample for AP. In-line SPE-CE/MS is useful for the screening of MA in hair because MA in a single 1-cm hair strand is detectable at the concentration of 1.6 ng/mg hair.
Top-cited authors
Kazumasa Sekiguchi
  • National Institute of Police Science Japan
Shinichi Suzuki
  • National Institute of Police Science Japan
Noriyoshi Takasawa
  • Edogawa University
Kazuhiko Imaizumi
  • National Institute of Police Science Japan
Ritsuko Sugita
  • National Research Institute of Police Science Japan