In Japan, possession of germinable cannabis seeds for cultivation purposes is subject to prosecution. Cannabis seeds are marketed after being treated to prevent germination (heated or crushed). Currently, forensic examination of cannabis seeds is conducted by cultivating the seeds after germination tests for several weeks and then performing morphological observation and chemical analysis on the true leaves. In this study, we attempted to construct a rapid and simple method for the identification of cannabis seeds by combining the color reaction using 2,3,5-Triphenyl-2H-tetrazolium Chloride (TTC), a reagent that discriminates between living and dead cells, and DNA testing using a commercially available simple kit.
The color reaction using TTC can determine the viability of peeled embryos within 20 min at 45 ℃ as previously reported. This method is effective for quickly determining whether a seed has been heat-treated or not. However, in the color reaction, a commercial health food seed that claimed to be unheated showed some coloration. This sample had been crushed to prevent germination and was easily identified as non-germinable by morphological examination. After the color reaction, the embryos could be directly used for DNA extraction without washing, and the DNA testing could be carried out in about 2 hours by following the instruction manual of the kit. By following the above procedure, it was possible to identify in one day whether a seed was a germinable cannabis or not, without the need to cultivate the plant. This method is expected to make a significant contribution to improving the efficiency of cannabis seed analysis.
Recently, AXO Science released STK Sperm Tracker Lab (STK sheet) and STK Sperm Tracker SPRAY (STK spray) to identify human semen stains. In this study, we examined the STK sheet and STK spray for their specificity to detect human semen, sensitivity, effects on DNA, and response to old semen samples. Additionally, STK sheet was examined for optimal crimping time depending on the type of fabric. STK spray was examined through assessing the effect of ultraviolet (UV) radiation after spraying. As a result of this study, STK sheet and STK spray were found to exhibit high specificity for the detection of human semen. These reagents did not affect the yield of extracted DNA or its degradation. STK sheet and STK spray can detect semen up to 1/25th and 1/100th dilutions, respectively. Both STK sheet and STK spray can react with semen samples left at room temperature for over a year. We found that the crimping time on STK sheet differs depending on the type of fabric, and the exposure to UV radiation for an hour after using STK spray does not affect DNA degradation. Conclusively, both STK sheet and STK spray are suitable to identify the human semen stains.
Human DNA quantification is an important step for subsequent short tandem repeat (STR) analysis and interpretation because it can provide various information on the quantity and quality for the extracted DNA. In this study, we compared four commercially available quantification kits based on a TaqMan assay and an intercalating-dye based quantification kit. The following three points were investigated: 1) variations of the average peak heights among individuals when 1 ng of DNA, the amount being determined by each quantification kit, was amplified using GlobalFiler and Yfiler Plus PCR Amplification Kits, 2) effect of the presence of a female DNA on the quantification of male DNA, and 3) relationship between “Degradation Index” and the STR electropherograms. The average peak heights generated using GlobalFiler showed less individual-dependent variations in the four TaqMan based kits than the intercalating-dye based kit. The average peak heights generated using Yfiler Plus showed similar variations among the three TaqMan based kits capable of male DNA quantification along with total human DNA. The presence of a large amount of female DNA had little effect on male DNA quantification in all the three kits. When highly degraded DNA samples were quantified, the “Degradation Indices” differed significantly among three kits. It was probably due to the different amplicon sizes of the long fragment targets among kits. Before implementing a new human DNA quantification kit in casework, it is essential to understand the characteristics of the adopted quantification kit.
Age estimation by use of DNA methylation rate have recently been one of the hottest topics in forensic genetics, thus many age prediction models have been developed. The age prediction model developed by Bekaert et al was evaluated as the best method comparing six blood-based models in 2019. In this study, Bekaert's age estimation model was validated with 143 Japanese blood samples (98 living and 45 dead blood samples). The difference between duplicate measurement of DNA methylation rate with pyrosequencer was less than one-year per one-gene in age prediction result. Overall mean absolute error (MAE) was 3.24, and root mean square error (RMSE) was 4.13. MAE of the elder group was larger than that of the young group. No statistically significant difference in absolute prediction error was observed between living and deceased or male and female blood samples. Furthermore, a novel model was developed with support vector regression. The MAE and RMSE were 3.49 and 4.51 for test set, respectively. In total, 200 ng of pre-bisulfite-treated DNA was essential to perform these predictions. To reduce the required amount of DNA, new models based on only ELOVL2 methylation rate requiring only 40 ng of DNA were also developed. The MAE and RMSE were 3.15 and 4.20 for test set, respectively. These results suggest that both Bekaert's age estimation model and our novel model have great potential in estimating one's age on forensic practices with some points to note.
Species identification of botanical and fungal evidences offer beneficial information in crime investigation. As these biological species are highly diverse and complex, their DNA sequence analysis is effective in the field of forensic science. In this study, the internal transcribed spacer (ITS) region on the ribosomal DNA of plant and fungal samples was examined as a targeting locus. For 30 plant and 9 fungal samples, direct sequencing was performed for both ITS1 and ITS2 region using modified universal primers and M13 tag primers. Each resulting sequence was evaluated by a nucleotide BLAST homology search. The top hits for sequence homology against each sample matched the actual species with almost 100% identity. However, homology search does not always result in sequence similarity/identity among closely-related species; therefore, results of homology search must be carefully assessed.
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.
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.
We have developed a quantitative method that uses GC-FID and QuEChERS to measure Δ-9-tetrahydrocannabinol (Δ9-THC) in cannabis-infused foods. Reported methods for the analysis of cannabis-infused foods typically use liquid chromatography, but obtaining Δ9-THC used in these methods, with the certified concentration, is difficult in Japan. Therefore, we attempted to apply a method, obtained from the United Nations Office on Drugs and Crime, that allows the quantitative analysis of Δ9-THC in herbal cannabis, hashish, and liquid using cannabinol as the reference material. In this study, the QuEChERS method, which is widely used in food analysis, was adopted. The EN 15662 salt method (EN salt method) and Enhanced Matrix Removal-Lipid method (EMR-Lipid method) were compared using recovery tests for Δ9-THC added to four types of food samples. The EN salt method was found to be superior to the EMR-Lipid method for some foods. A similar test was conducted on six different types of foods using the EN salt method. The recovery was 95.2% to 113.5%. The precision was tested at two levels of Δ9-THC content in three trials over three days. The intra-day precision was 9.8% and 1.4% for 0.1 mg/g and 1 mg/g Δ9-THC, respectively. The inter-day precision was 16.6% and 4.4% for 0.1 mg/g and 1 mg/g Δ9-THC, respectively. Cannabis-infused brownie was homogenized, divided, and tested in three laboratories. The average values of Δ9-THC content at the three laboratories were 0.33, 0.43, and 0.48 mg/g. We analyzed Δ9-THC content in 13 cannabis edibles seized in Japan. The overall Δ9-THC content ranged from 0.33 mg/g to 7.14 mg/g, and the content per piece ranged from 4.3 mg to 13.9 mg. This study demonstrates a useful method for the quantification of Δ9-THC in cannabis-infused foods.
Time-dependant change in compositions of ignitable liquid residues (ILRs) from fire debris samples prepared from three common flooring materials (i.e., wood flooring, carpet on wood flooring, and tatami) burned with gasoline or kerosene were examined, and their detectable periods were evaluated in gas chromatography with mass spectrometry (GC/MS) analysis with NeedlEx®.
The results revealed that the type of flooring material affected the compositions and evaporation rates of each ILR. Whereas ILRs from debris burned with gasoline showed different compositions from a simply evaporated gasoline reference, ILRs from debris burned with kerosene showed considerable similarity to the evaporated kerosene reference. The detectable periods of dynamic headspace extraction revealed in GC/MS analysis with NeedlEx® also far exceeded the periods of detector tubes.