Discrimination of Camellia cultivars using iD-NA analysis.

Recently, many new cultivars have been taken abroad illegally, which is now considered an international issue. Botanical evidence found at a crime scene provides valuable information about the origin of the sample. However, botanical resources for forensic evidence remain underutilized because molecular markers, such as microsatellites, are not available without a limited set of species. Multiplexed intersimple sequence repeat (ISSR) genotyping by sequencing (MIG-seq) and its analysis method, identification of not applicable (iD-NA), have been used to determine several genome-wide genetic markers, making them applicable to all plant species, including those with limited available genetic information. Camellia cultivars are popular worldwide and are often planted in many gardens and bred to make new cultivars. In this study, we aimed to analyze Camellia cultivars/species through MIG-seq. MIG-seq could discriminate similar samples, such as bud mutants and closely related samples that could not be distinguished based on morphological features. This discrimination was consistent with that of a previous study that classified cultivars based on short tandem repeat (STR) markers, indicating that MIG-seq has the same or higher discrimination ability as STR markers. Furthermore, we observed unknown phylogenetic relationships. Because MIG-seq can be applied to unlimited species and low-quality DNA, it may be useful in various scientific fields.

Simultaneous determination of eight catechins and four theaflavins in bottled tea by liquid chromatography-tandem mass spectrometry for forensic analysis.

Tea, and particularly bottled tea, is widely consumed worldwide and is often encountered at crime scenes in poisoning cases or used in place of urine in drug abuse monitoring. Tea is a rich source of polyphenols, such as catechins and theaflavins, and these compounds are useful for identification of trace quantities of tea samples. However, information on the contents of catechins and theaflavins in bottled tea is limited. In this study, a method was developed for simultaneous analysis of eight catechins and four theaflavins in tea using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentrations of these polyphenols were determined in bottled black, oolong, and green teas after a simple pretreatment process by the standard addition method. The developed LC-MS/MS method was rapid and all tested polyphenol compounds were separated within ~14 min. All tea types contained all the catechins, at varying concentrations, but not all the theaflavins were present in all the tea types. This indicates that the theaflavin composition reflects the degree of the fermentation and could be used for discrimination among different types of tea. All the green tea samples contained all eight catechins; however, the concentrations of these compounds varied among the tea samples. Principal component analysis and hierarchical cluster analysis were useful for discrimination of samples. It has been unclear whether the variations of chemical components are useful for forensic discrimination. Our results demonstrate that, in addition to identification of tea varieties, catechins and theaflavins can be used for the discrimination of bottled tea samples.

Semi-automated scanning electron microscopy energy dispersive X-ray spectrometry forensic analysis of soil samples.

Soil is commonly analyzed to provide evidence because soil particles adhere to almost everything that may be of forensic interest. Particles derived from soil can be quantitatively analyzed by scanning electron microscopy with energy dispersive X-ray spectrometry (SEM-EDS). We developed a semi-automated SEM-EDS method for analyzing soil minerals. Soil was analyzed using the SEM-EDS automated particle analysis program, then the mineral species were identified from their chemical components. Chi-squared tests were used to discriminate between different minerals, and these tests were also applied to soil samples. The method put samples with the same parent population into the same group and discriminated between samples with different parent populations even when 26 particles were analyzed. The method successfully identified most soil samples with different parent populations as different. The results proved that the method could be used in forensic cases and provide new insights into the forensic analysis of soil samples.

Species identification of white false hellebore (Veratrum album subsp. oxysepalum) using real-time PCR.

Food poisoning is frequently caused by the accidental ingestion of toxic plants that possess strong morphological similarities to edible plants. False helleborine (Veratrum album) is one of the most common plants involved in such accidents. In cases of poisoning by toxic plants, rapid and accurate identification, usually based on the morphological or chemical analysis of plant parts, is required for appropriate medical treatment or forensic investigation. However, morphological examinations require experience in systematic botany because the samples are fragmentary, and chemical analysis of natural compounds can be difficult. In this study, we developed a TaqMan real-time PCR method using trnH-psbA and trnL-trnF that could be carried out in 30-60min. The lower detection limit was less than 10pg of DNA and the primer sets were specific to V. album and Veratrum stamineum. Mixed samples, cooked samples, and simulated gastric contents were successfully identified, and a multiplex assay of two regions was also possible. These results indicate that the TaqMan real-time PCR analysis is a very effective method to detect small samples of V. album and V. stamineum accurately and rapidly in poisoning cases.

Real-Time PCR Quantification of Chloroplast DNA Supports DNA Barcoding of Plant Species.

Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.

Forensic DNA Analysis of Wheat Flour as Commonly Used in White Powder Cases.

In the wake of terrorist attacks using anthrax and ricin, white powder is often encountered in cases of malicious mischief and terrorist threats. Wheat flour is a common white powder encountered in such criminal investigations. We used DNA analysis to investigate wheat flour samples for identification and discrimination as trace evidence. Species identification of commercially available wheat flour was carried out by sequencing a partial region of the ribulose bisphosphate carboxylase large subunit gene (rbcL). Samples were discriminated using short tandem repeat (STR) analysis. The rbcL sequences of all wheat flour samples were identical and showed a high level of similarity to known wheat (Triticum aestivum L.) sequences. Furthermore, flours had characteristic patterns in STR analyses, with specific cultivars showing distinctive patterns. These results suggested that the identification of wheat flour species is possible using rbcL sequencing, and that STR analysis is useful for discriminating between samples.

Rapid discrimination of Legionella by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Molecular typing is an important tool in the surveillance and investigation of human Legionella infection outbreaks. In this study, two molecular typing methods, pulsed-field gel electrophoresis (PFGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), were used to discriminate 23 Legionella pneumophila strains. The usefulness of MALDI-TOF-MS was demonstrated. The MALDI-TOF-MS fingerprinting with filtered small acid-soluble molecules gave different molecular profiles among strains, and the clustal analysis with MALDI-TOF-MS showed a high discrimination of strains the same as that with PFGE. In addition, MALDI-TOF-MS data could be generated within a few hours after the initial culture, although PFGE analyses took several days to complete. Thus, MALDI-TOF-MS offers a simple and rapid discrimination technique that could aid in the tracking of fast-spreading outbreaks of Legionella.

Potential utility of DNA sequence analysis of long-term-stored plant leaf fragments for forensic discrimination and identification.

This study examined the potential utility of DNA sequence analysis to discriminate and identify plant material in forensic investigations. DNA was extracted from plant leaf fragments of 11 species stored for 5 to 22 years after collection. The trnH-psbA intergenic spacer and 316 bp of the rbcL gene were successfully amplified and sequenced for all fragments except for the trnH-psbA spacer of one sample. All of the plant samples were discriminated in pairwise comparisons of the sequences. Using a combination of local and global genetic databases is likely to provide greater reliability in search results to identify forensic samples from sequence data.

Characterization of the catalytic activity of the gamma-phage lysin, PlyG, specific for Bacillus anthracis.

Bacillus anthracis causes anthrax, a lethal disease affecting humans that has attracted attention due to its bioterrorism potential. PlyG is a lysin of gamma-phage, which specifically infects B. anthracis and lyses its cell wall. PlyG contains a T7 lysozyme-like amidase domain, which appears to be the catalytic domain, in the N-terminal region and has a high degree of sequence similarity with PlyL, which is an N-acetylmuramoyl-l-alanine amidase encoded by the B. anthracis genome. Here, we demonstrated that two amino acid residues of PlyG, H29 and E90, are necessary for its catalytic activity in B. anthracis. These residues are structurally analogous to residues whose mutation in T7 lysozyme abolished its catalytic activity. A C-terminal deletion mutant of PlyG lacking the core sequence for binding to B. anthracis showed completely abolished binding activity, unlike PlyL, despite high sequence similarity with PlyL in the N-terminal region. This suggests that the C-terminal binding domain, as well as the N-terminal catalytic domain, is essential for the catalytic activity of PlyG. Our observations provide new insights into the mechanism of specific catalysis of PlyG in B. anthracis and may contribute to the establishment of new methods for anthrax therapy.

Forensic discrimination of sheet glass exposed to high temperature by the determination of trace impurities using ICP-MS.

The effect of heating on the refractive index (RI) and trace elemental compositions of glass was investigated in order to develop an accurate discrimination method of glass fragments exposed to the high temperature of fire on illegal entrance into a crime scene for robbery. Fragments taken from 5 different sheet glasses were subjected to RI measurement and analysis of trace elements using ICP-MS before and after heating at 764 degrees C for 2 min. The difference in the RI between the heated and non-heated fragments ranged from 0.0012 to 0.0015, which corresponds to 6 times more than the variation of the RI within a pane of glass. In contrast, profiles of 10 elements (Co, Rb, Sr, Zr, Mo, Ba, La, Ce, Nd and Pb) in glass exhibited no significant difference between the heated and non-heated ones. In conclusion, the forensic discrimination of glass fragments must be performed not by RI measurement, but by analysis of the elemental compositions when glass evidence could be exposed to the high temperature of fire.