Using rDNA ITS2 barcoding to identify kratom (Mitragyna speciosa) from the genus Mitragyna and Neolamarckia cadamba.

This study collected 80 samples of suspected kratom plant powder. A polymerase chain reaction sequence analysis was conducted using two sets of DNA barcode primers for plant ribosomal (r)DNA internal transcribed spacers (ITSs), namely, ITS3/ITS4 and ITS-p3/ITS-u4. Among the 80 samples, 40 were analyzed using the ITS3/ITS4 primer pair, and then DNA sequences were subjected to a National Center for Biotechnology Information-Basic Local Alignment Search Tool (NCBI-BLAST) comparison. Results showed that 29 samples had a 100% match (364/364) with Mitragyna speciosa (kratom), and 6 samples had a 99.73% match (363/364) with M. speciosa, whereas 5 samples had disordered and unreadable sequences. The 5 unreadable samples and an additional 40 suspected kratom samples were then analyzed using the ITS-p3/ITS-u4 primer pair, followed by an NCBI-BLAST comparison. Among these, 32 samples had a 100% match (404/404) with M. speciosa, and 11 samples had a 99.75% match (403/404) with M. speciosa. Among the samples with sequences matching M. speciosa, three distinct types were observed (no variance/404, 287M/404, and 287A/404). One sample had a 99.51% match (404/406) with Neolamarckia cadamba, and another sample had a sequencing length of 305 bp, with 25 positions showing mixed base pairs, indicating a mixture of different species. Analysis of the mixed base pair pattern suggested a possible mixture of M. speciosa and N. cadamba. Actually, M. speciosa and N. cadamba have very similar external morphologies. This indicates that the ITS-p3/ITS-u4 primer pair is effective in distinguishing mixtures of M. speciosa and N. cadamba and is thus more suitable than ITS3/ITS4 for identifying and analyzing samples of suspected kratom plant powder.

Spermatozoa identification by the 3-plex MSRE-PCR assay: a collaborative exercise.

Identification of semen and spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. In cases of alleged sexual assault where there is a long time gap between the incident and sample collection, or in cases of low sperm count, current methods have limitations of specificity, in the case of presumptive tests for semen, or the problem of recording spermatozoa by microscopy if they are few in number. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) assay using a spermatozoa-specific DNA methylated marker to identify spermatozoa has been reported previously by our laboratory. A key advantage over current methods is the increased sensitivity and specificity. A transition from a research tool to operational use requires blind trial testing and inter-laboratory trials. We report on a collaborative exercise where reagents of the 3-plex MSRE-PCR were sent to six participating laboratories. Each laboratory used their own equipment, consumables, and the presumptive reagents conventionally for body fluid (such as acid phosphatase or PSA), DNA extraction, and quantification in practical casework. The reagents and protocol for the 3-plex MSRE-PCR assay and 9 samples were provided by the organizing laboratory. The participating laboratories were requested to fill in the questionnaire after testing. The reported results from all the six participating laboratories were concordant and the expected correct results for the presence of spermatozoa. These outcomes verified the reproducibility and feasibility of the 3-plex MSRE-PCR assay. The results also indicated that the 3-plex MSRE-PCR assay was readily accessible to forensic laboratories for integrating it into current forensic casework processes.

Genetic polymorphisms of 12 X-chromosomal STR loci in Taiwanese individuals and likelihood ratio calculations applied to case studies of blood relationships.

Allele frequencies for the 12 short tandem repeat loci of the Investigator Argus X-12 kit were obtained from 514 unrelated Taiwanese individuals (327 males and 187 females). Hardy-Weinberg equilibrium tests with samples demonstrated no significant deviation from expected values for all 12 loci (p > 0.05). The linkage disequilibrium for the 12 loci in the female samples was identical to what was observed in other Han Chinese populations, with only the DXS10103 and DXS10101 loci showing significant linkage disequilibrium after corrected by Bonferroni's correction for multiple testing (p < 0.05/66). No significant differences were observed by population pairwise genetic distance analysis between Taiwanese and other Han Chinese populations. When compared with other Asian, European, and African populations, however, significant differences were observed at more than one locus. The combined mean exclusion chance was 0.99999 in duo cases and 0.99999999 in trio cases. This study used mathematical logic inferred likelihood ratio calculation formulas for full-sister, half-sister from the same father, and paternal grandmother-granddaughter relationships. The results for these three real familial cases suggest that these 12 short tandem repeat loci may appropriate for forensic relationship testing.

Y-chromosomal STRs haplotypes in the Taiwanese Paiwan population.

The distribution of Y-chromosomal short tandem repeat (Y-STR) haplotypes was determined in a population of Taiwanese Paiwan aboriginals. Using 17 Y-STR markers, a total of 135 haplotypes were observed, 102 of which were unique. The overall haplotype diversity for the 17 Y-STR loci tested was 0.9922 and the discrimination capacity was 0.6490. In addition, three novel intermediate alleles at the DYS448 locus were also found.

Genetic polymorphisms of 17 Y-chromosomal short tandem repeat loci in Atayal population of Taiwan.

AIM: To define the Y-chromosomal genetic structure in a sample of Atayal men from Taiwan. METHODS: Buccal swab samples were collected from 170 unrelated healthy male volunteers from Taiwanese aboriginal Atayal population. Genomic DNA was extracted and 17 Y chromosome-specific short tandem repeat loci (DYS456, DYS389I, DYS390, DYS389II, DYS458, DYS19, DYS385a/b, DYS393, DYS391, DYS439, DYS635, DYS392, Y GATA H4, DYS437, DYS438, and DYS448) were analyzed using the AmpFlSTR Yfiler Polymerase Chain Reaction Amplification Kit. RESULTS: A total of 99 different haplotypes were identified, 69 (69.7%) of which were unique. Total haplotype diversity was 0.9887. The most common haplotype was shared by 9 individuals in the study sample. Gene diversities ranged from 0.0574 for DYS438 to 0.6749 for DYS456. CONCLUSION: Our results will help provide the molecular genetic evidence for human settlement of the Pacific.

Asian online Y-STR Haplotype Reference Database.

For several years Y-chromosomal microsatellites (short tandem repeats, STRs) have been well established in forensic practice. In this context, the genetic characteristics of the Y chromosome (i.e. its paternal inheritance and lack of recombination) render STRs particularly powerful. However, genetic differences between male populations appear to be larger for Y-STRs than for autosomal STRs, a fact that is most likely due to the higher sensitivity of Y-chromosomal lineages to genetic drift (Forensic Sci Int 118 (2001) 153). The assessment of probabilities for matches between haplotyped male persons or traces/persons requires the typing of a large number of haplotypes in the appropriate reference populations. The haplotype data of a large number of European as well as South and North American populations have been collected and are continuously published online (Y-STR Haplotype Reference Database--YHRD; http://www.ystr.org). The most recent multicentric effort has led to the establishment of an Asian YHRD (http://www.ystr.org/asia) which has been available since January 2002. All databases are maintained and curated at the Institute of Legal Medicine, Humboldt-University, Berlin and will soon be fused to a global repository including populations from all continents.

Population data on X chromosome short tandem repeat loci HPRTB and AR in Taiwan.

This report contains the results of population studies on the X chromosome STR HPRTB and AR carried out in Taiwan. The numbers of unrelated individuals were 428: female 143 and male 285 for HPRTB locus, and 416: female 142 and male 274 for AR locus.