A grass molecular identification system for forensic botany: a critical evaluation of the strengths and limitations.

Plant material is frequently encountered in criminal investigations but often overlooked as potential evidence. We designed a DNA-based molecular identification system for 100 Australian grasses that consisted of a series of polymerase chain reaction assays that enabled the progressive identification of grasses to different taxonomic levels. The identification system was based on DNA sequence variation at four chloroplast and two mitochondrial loci. Seventeen informative indels and 68 single-nucleotide polymorphisms were utilized as molecular markers for subfamily to species-level identification. To identify an unknown sample to subfamily level required a minimum of four markers or nine markers for species identification. The accuracy of the system was confirmed by blind tests. We have demonstrated "proof of concept" of a molecular identification system for trace botanical samples. Our evaluation suggests that the adoption of a system that combines this approach with DNA sequencing could assist the morphological identification of grasses found as forensic evidence.

A Cannabis sativa STR genotype database for Australian seizures: forensic applications and limitations.

A genetic database was established with the aim of documenting the genetic diversity of Cannabis sativa in Australia for future utilization in forensic investigations. The database consisted of genotypes at 10 validated short tandem repeat loci for 510 plants representing drug seizures from across Australia and 57 fiber samples. A total of 106 alleles and 314 different genotypes were detected. All fiber samples exhibited unique genotypes while 55% of the drug samples shared a genotype with one or more samples. Shared genotypes were mostly found within seizures; however, some genotypes were found among seizures. Statistical analysis indicated that genotype sharing was a consequence of clonal propagation rather than a lack of genetic resolution. Thus, the finding of shared genotypes among seizures is likely due to either a common supplier, or direct links among seizures. Notwithstanding the potential intelligence information provided by genetic analysis of C. sativa, our database analysis also reveals some present limitations.

Developmental validation of a Cannabis sativa STR multiplex system for forensic analysis.

A developmental validation study based on recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM) was conducted on a multiplex system of 10 Cannabis sativa short tandem repeat loci. Amplification of the loci in four multiplex reactions was tested across DNA from dried root, stem, and leaf sources, and DNA from fresh, frozen, and dried leaf tissue with a template DNA range of 10.0-0.01 ng. The loci were amplified and scored consistently for all DNA sources when DNA template was in the range of 10.0-1.0 ng. Some allelic dropout and PCR failure occurred in reactions with lower template DNA amounts. Overall, amplification was best using 10.0 ng of template DNA from dried leaf tissue indicating that this is the optimal source material. Cross species amplification was observed in Humulus lupulus for three loci but there was no allelic overlap. This is the first study following SWGDAM validation guidelines to validate short tandem repeat markers for forensic use in plants.

Gamma-hydroxybutyrate poisoning from toy beads.

A 2-year-old boy and a 10-year-old girl presented to the emergency department with a decreased level of consciousness. The girl had had persistent vomiting and a seizure. Urine metabolic screening tests were positive for gamma-hydroxybutyrate (GHB). Samples from toy beads ingested by both children contained 1,4-butanediol, which is metabolised to GHB in humans. Regulatory authorities were notified, leading to an international recall of the toy beads.

Unintentional paediatric ingestions of angiotensin converting enzyme inhibitors and angiotensin II receptor antagonists.

AIMS: There is limited information on safety of angiotensin converting enzyme (ACE) inhibitors and angiotensin II (AII) receptor antagonists in unintentional paediatric ingestions. This study was conducted with the aim of developing referral guidelines for poison information centres. METHODS: Calls to the NSW poison information centre from January 2002 to July 2004 regarding paediatric ingestion of ACE inhibitors and AII receptor antagonists were recruited and prospectively followed up. Information collected included: demographics (age, gender, weight), type of exposure (unintentional, therapeutic error), ingested dose and clinical effects. Dose was reported in defined daily doses (DDD) to compare across and within the two drug classes with respect to the normal adult dose. RESULTS: Nineteen cases of paediatric ingestion of ACE inhibitors and AII receptor antagonists were included. The median age was 2 years (Interquartile range (IQR): 20-33 months) and the median dose ingested was 1 DDD (IQR: 1-2). There were nine ACE inhibitor ingestions and 10 AII receptor antagonist ingestions. One of nine children (11%) observed in hospital developed transient hypotension but required no treatment and recovered without complication. This child ingested an ACE inhibitor and ingested >3 DDD. CONCLUSION: Unintentional paediatric ingestions of ACE inhibitors and AII receptor antagonists resulted in the majority of children remaining asymptomatic. ACE inhibitor ingestions under 2 DDD can be observed at home provided the child is asymptomatic and there is a responsible adult to observe the child. The dose required for observation in AII receptor antagonist ingestions is less clear.

Organelle DNA haplotypes reflect crop-use characteristics and geographic origins of Cannabis sativa.

Comparative sequencing of cannabis individuals across 12 chloroplast and mitochondrial DNA loci revealed 7 polymorphic sites, including 5 length variable regions and 2 single nucleotide polymorphisms. Simple PCR assays were developed to assay these polymorphisms, and organelle DNA haplotypes were obtained for 188 cannabis individuals from 76 separate populations, including drug-type, fibre-type and wild populations. The haplotype data were analysed using parsimony, UPGMA and neighbour joining methods. Three haplotype groups were recovered by each analysis method, and these groups are suggestive of the crop-use characteristics and geographical origin of the populations, although not strictly diagnostic. We discuss the relationship between our haplotype data and taxonomic opinions of cannabis, and the implications of organelle DNA haplotyping to forensic investigations of cannabis.

Lignocaine and chlorhexidine toxicity in children resulting from mouth paint ingestion: a bottling problem.

BACKGROUND: A pharmaceutical product was marketed in Australia for 'teething' in an almost identical container to a popular paediatric paracetamol preparation. The product contained lignocaine and chlorhexidine. The similarity of the packaging resulted in large number of therapeutic errors in which the 'teething' preparation was given in error for paracetamol. As the exact dose of the erroneously administered mouth paint was known this provided an opportunity for outcome assessment of lignocaine ingestion. METHODS: Calls to two state poison information centres regarding this product were prospectively followed up. Information collected included: demographics, type of exposure, details of the exposure and adverse effects. A systematic review of the literature was used to identify all previous reported cases of lignocaine and chlorhexidine ingestion. RESULTS: There were 28 cases with complete follow up where the product was given in therapeutic errors (10 girls and 18 boys; median age 11 months; range 2 months-4 years). The mean ingested dose of lignocaine was 2.7 mg/kg (standard deviation 1.3 mg) and chlorhexidine was 0.06 mg/kg (standard deviation 0.03 mg). The largest ingested lignocaine dose was 5.9 mg/kg. Two children developed minor symptoms: one vomited twice and the other was reported to have increased salivation and difficulty with solid food for 20 min. No other adverse effects were reported. The literature review suggested that severe effects occurred with doses more than 15 mg/kg. CONCLUSION: No major adverse effects occurred with lignocaine ingestions of less than 6 mg/kg and it would be appropriate to observe these patients at home. Chlorhexidine did not appear to cause clinical effects in this low concentration.

Short tandem repeat (STR) DNA markers are hypervariable and informative in Cannabis sativa: implications for forensic investigations.

Short tandem repeat (STR) markers are the DNA marker of choice in forensic analysis of human DNA. Here we extend the application of STR markers to Cannabis sativa and demonstrate their potential for forensic investigations. Ninety-three individual cannabis plants, representing drug and fibre accessions of widespread origin were profiled with five STR makers. A total of 79 alleles were detected across the five loci. All but four individuals from a single drug-type accession had a unique multilocus genotype. An analysis of molecular variance (AMOVA) revealed significant genetic variation among accessions, with an average of 25% genetic differentiation. By contrast, only 6% genetic difference was detected between drug and fibre crop accessions and it was not possible to unequivocally assign plants as either drug or fibre type. However, our results suggest that drug strains may typically possess lower genetic diversity than fibre strains, which may ultimately provide a means of genetic delineation. Our findings demonstrate the promise of cannabis STR markers to provide information on: (1) agronomic type, (2) the geographical origin of drug seizures, and (3) evidence of conspiracy in production of clonally propagated drug crops.