Cannabis Seeds Authentication by Chloroplast and Nuclear DNA Analysis Coupled with High-Resolution Melting Method for Quality Control Purposes.

Background: Cannabis plants and their seed have been used in many cultures as a source of medicine and feeding during history. Today, there is an increasing demand for cannabis seeds for medical use. Moreover, a seed sales market with no legal regulations has also grown. This may pose some issues if a quality control is not set in place. Identification of cannabis strains is important for quality control purposes in a nonregulated growing market and in cases of illegal traffic and medical use. Owing to the high price as a pharmacological drug, commercial products of cannabis plants and seeds for medical users are often subjected to adulterations, either when packing or distributing certified seeds in the market. Materials and Methods: Cannabis commercial seeds and cannabis seeds for medical use were analyzed with high-resolution melting (HRM) analysis using barcoding markers. Humulus lupulus L. plants from a local market were used as outgroup control. DNA barcoding uses specific regions of the genome to identify differences in the genetic sequence of conserved regions such as internal transcribed spacer (ITS) and rbcL. DNA barcoding data can be generated with real-time polymerase chain reaction combined with HRM analysis to distinguish specific conserved DNA regions of closely related species. HRM analysis is the method of choice for rapid analysis of sequence variation. Results: The melting temperature (Tm) of homogeneous packages was consistent with single genotypes. However, packages containing contaminating seeds showed Tm differences of 0.2°C on average. Conclusions: An effective, rapid, and low-cost method based on ITS nuclear DNA and on chloroplast rbcL regions for screening and detection of contamination in commercial cannabis seeds was developed and applied for the analysis of different samples. This approach can be used as a quality control tool for cannabis seeds or other plant material.

ITS barcoding using high resolution melting analysis of Cannabis sativa drug seizures in Chile: A forensic application.

Cannabis sativa L. is a plant cultivated worldwide as a source of fiber, medicine, and intoxicant. Traditionally, C. sativa is divided into two main types: fiber type (hemp) and drug type. Drug-type C. sativa differs from hemp by the presence of a high quantity of the psychoactive drug, Δ9-tetrahydrocannabinol (Δ9 THC). Cannabis sativa is the most commonly used used illicit controlled substance in Chile. Chile is the third greatest consumer of Cannabis in South America. The objective of this study was to determine the genetic composition of ten drug seizures of Cannabis spp. in the south of Chile using a high resolution melting (HRM) strategy combined with a barcoding marker, ITS. C. sativa samples were selected from previously processed more than a thousand crime cases at the, Araucania region crime lab, National Dept. of Health. Ten cases were selected. Sample collection was based on the following: a) dry and fresh samples with no evidence of decomposition or degradation, b) defined plant fragments such as flowers and leaves from individual plants and, c) samples with different content of THC, CBN and CBD. Five sub samples were randomly selected from each case (N=50). The commercial Silver Haze strain was used as a control. Two real-time PCR and HRM analyses were conducted. The first analysis was performed with a representative sample of each of the 10 cases studied. Then a second assay was performed with all subsamples of cases 1, 5 and 8. Results showed that real-time PCR combined with HRM analysis using ITS allowed to determine the genetic composition of cannabis in all cases studied. The derivative of melting and the analysis of the shape of the curve and the peak of Tm, showed that three groups can be clearly distinguished. A first group exhibited a peak of Tm close to 87.4°C and includes cases 7 and 8. A second group had a peak of Tm close to 87.6°C and includes case 5. A third group displayed a peak of Tm close to 87.9°C and includes case 1, 6 and Silver Haze strain. A second experiment was performed using subsamples of cases 1, 5 and 8. Case 1 displayed a unique composition of the drug suggesting that this seizure contained cannabis clonally propagated. In case 5, two genotypes were present, therefore this could be associated with two strain or two different origin. Case 8, was composed of a mixture of cannabis strains indicating the presence of various crop type and/or different biogeographic origin. In general, our results suggested genetically homogeneous seizures from Araucanía Region. The high latitude (37° 35' and 39° 37' South latitude) and the natural geographic borders that surround southern Chile helps the control of cannabis traffic into the country. Finally, HRM analysis coupled with the barcode ITS demonstrated to be a rapid and low-cost screening method.

Use of Eucalyptus DNA profiling in a case of illegal logging.

Eucalyptus is grown world-wide for paper pulp, solid wood, and other industries. Theft or illegal cutting of the trees causes hardship to owners of plantations and countries whose economies rely on the sale and export of eucalyptus products. Unfortunately, many of these crimes go unpunished due to lack of forensic evidence. Over 1200 short tandem repeat (STR) markers have been identified in the genomes of genus Eucalyptus and related species. However, their importance and utility in aiding forensic investigations of wood theft have not been explored. This study evaluated nine STRs for diversity and applied them to a case involving suspected wood theft. As expected, three dinucleotide STR markers showed greater variability but resulted in harder to interpret profiles. Four STR tetranucleotide markers evaluated in this study were found to contain additional repeat structures (dinucleotide or trinucleotide) that enhanced their variability but resulted in profiles with peaks at multiple stutter positions and heterozygote peak imbalance. The most promising STR markers were EGM37 and EMBRA 1374. Though less variable, they yielded robust and reproducible DNA profiles. All nine STR markers were applied to a case involving suspected wood theft. Samples were collected from seized wood and from remaining stumps in a plantation. No DNA match was found, thus eliminating the evidence samples as having originated from the forest. Dendrochronology analysis also resulted in an exclusion. This case study represents the first report using STR markers in any eucalyptus species to provide DNA evidence in a case of suspected wood theft.

High-resolution melting of the cytochrome B gene in fecal DNA: A powerful approach for fox species identification of the Lycalopex genus in Chile.

Easy, economic, precise species authentication is currently necessary in many areas of research and diagnosis in molecular biology applied to conservation studies of endangered species. Here, we present a new method for the identification of three fox species of the Lycalopex genus in Chile. We developed an assay based on high-resolution melt analysis of the mitochondrial cytochrome B gene, allowing a simple, low cost, fast, and accurate species determination. To validate the assay applicability for noninvasive samples, we collected fecal samples in the Atacama Desert, finding unexpectedly one species outside of its known distribution range. We conclude that the assay has a potential to become a valuable tool for a standardized genetic monitoring of the Lycalopex species in Chile.

Hybrid identification in Nothofagus subgenus using high resolution melting with ITS and trnL approach.

The genus Nothofagus is the main component of southern South American temperate forests. The 40 Nothofagus species, evergreen and deciduous, and some natural hybrids are spread among Central and Southern Chile, Argentina, New Zealand, Australia, New Guinea and New Caledonia. Nothofagus nervosa, Nothofagus obliqua and Nothofagus dombeyi are potentially very important timber producers due to their high wood quality and relative fast growth; however, indiscriminate logging has degraded vast areas the Chilean forest causing a serious state of deterioration of their genetic resource. The South of Chile has a large area covered by secondary forests of Nothofagus dombeyi. These forests have a high diversity of species, large amount of biomass and high silvicultural potential. This work shows a case of hybrid identification in Nothofagus subgenus in different secondary forests of Chile, using high resolution melting. Unknown samples of Nothofagus subgenus are genetically distinguishable with the ITS region of Nothofagus antarctica, Nothofagus nitida and N. obliqua species. It was not possible to distinguish between unknown samples of Andean versus coastal origin. Melting curves with ITS approach of unknown material are genetically similar, positioned between N. dombeyi and N. antarctica and distant from N. nitida. The unknown samples are genetically very close to Nothofagus dombeyi. This suggests the presence of hybrid individuality between species (N. dombeyi × N. antarctica) with the possibility of introgression towards the gene pool of N. antarctica, producing the deciduous foliage that is both present. The trnL locus has no distinction between the N. dombeyi and N. antarctica species, since a similar melting curve is present and equal Tm (80.00 °C). The trnL locus cannot be genetically distinguished from one unknown sample of Nothofagus to another, as highlighted in this study.

Psychedelic fungus (Psilocybe sp.) authentication in a case of illegal drug traffic: sporological, molecular analysis and identification of the psychoactive substance.

In nature, there are >200 species of fungi with hallucinogenic properties. These fungi are classified as Psilocybe, Gymnopilus, and Panaeolus which contain active principles with hallucinogenic properties such as ibotenic acid, psilocybin, psilocin, or baeocystin. In Chile, fungi seizures are mainly of mature specimens or spores. However, clandestine laboratories have been found that process fungus samples at the mycelium stage. In this transient stage of growth (mycelium), traditional taxonomic identification is not feasible, making it necessary to develop a new method of study. Currently, DNA analysis is the only reliable method that can be used as an identification tool for the purposes of supporting evidence, due to the high variability of DNA between species. One way to identify the species of a distinctive DNA fragment is to study PCR products analyzed by real time PCR and sequencing. One of the most popular sequencing methods of forensic interest at the generic and intra-generic levels in plants is internal transcribed spacer (ITS). With real time PCR it is possible to distinguish PCR products by differential analysis of their melting temperature (Tm) curves. This paper describes morphological, chemical, and genetic analysis of mycelia of psychedelic fungi collected from a clandestine laboratory. The fungus species were identified using scanning electron microscopy (SEM), mass spectrometry, HRM analysis, and ITS sequencing. The sporological studies showed a generally smooth surface and oval shape, with maximum length 10.1 µm and width 6.4 µm. The alkaloid Psilocyn was identified by mass spectrometry, while HRM analysis and ITS sequencing identified the species as Psilocybe cubensis. A genetic match was confirmed between the HRM curves obtained from the mycelia (evidence) and biological tissue extracted from the fruiting bodies. Mycelia recovered from the evidence and fruiting bodies (control) were genetically indistinguishable.

Identification case of evidence in timber tracing of Pinus radiate, using high-resolution melting (HRM) analysis.

Fast, accurate detection of plant species and their hybrids using molecular tools will facilitate assessment and monitoring of timber tracing evidence. In this study the origin of unknown pine samples is determined for a case of timber theft in the region of Araucania southern Chile. We evaluate the utility of the trnL marker region for species identification applied to pine wood based on High Resolution Melting. This efficient tracing methods can be incorporated into forestry applications such as certification of origin. The object of this work was genotype identification using high-resolution melting (HRM) and trnL approaches for Pinus radiata (Don) in timber tracing evidence. Our results indicate that trnL is a very sensitive marker for delimiting species and HRM analysis was used successfully for genotyping Pinus samples for timber tracing purposes. Genotyping samples by HRM analysis with the trnL1 approach allowed us to differentiate two wood samples from the Pinaceae family: Pinus radiata (Don) and Pseudotsuga menziesii (Mirb.) Franco. The same approach with Pinus trnL wood was not able to discriminate between samples of Pinus radiata, indicating that the samples were genetically indistinguishable, possibly because they have the same genotype at this locus. Timber tracing with HRM analysis is expected to contribute to future forest certification schemes, control of illegal trading, and molecular traceability of Pinus spp.

Promoter methylation profile in gallbladder cancer.

BACKGROUND: Methylation in the promoter region of genes is an important mechanism of inactivation of tumor suppressor genes. Our objective was to analyze the methylation pattern of some of the genes involved in carcinogenesis of the gallbladder, examining the immunohistochemical expression of proteins, clinical features, and patient survival time. METHODS: Twenty cases of gallbladder cancer were selected from the frozen tumor bank. The DNA extracted was analyzed by means of a methylation-specific polymerase chain reaction test for the CDKN2A (p16), MLH1, APC, FHIT, and CDH1 (E-cadherin) genes. Morphological and clinical data and follow-up information were obtained. RESULTS: All cases were in an advanced stage: histologically moderate or poorly differentiated tumors (95%). Methylation of the promoter area of genes was observed in 5%, 20%, 30%, 40%, and 65% of cases, and an altered immunohistochemical pattern (AIP) in 5%, 35%, 21%, 25%, and 66% for the MLH1, CDKN2A, FHIT, APC, and CDH1 genes, respectively. The Kappa concordance index between methylation of the promoter area and AIP for the MLH1 and CDH1 genes was very high (K > 0.75) and substantial for APC (K > 0.45). No correlation was found between survival time and the methylation of the genes studied. CONCLUSIONS: The high frequency of gene methylation (with the exception of MLH1) and the high agreement between AIP and methylation of the gene promoter area for the MLH1, APC, and CDH1 genes suggest that the inactivation of tumor suppressor genes and of the genes related to the control of cellular proliferation through this mechanism is involved in gallbladder carcinogenesis.

[Promoter methylation profile in gastric cancer]. / Estudio del patrón de metilación génico en el cáncer gástrico en Chile.

BACKGROUND: Promoter genomic DNA methylation is an important inactivation mechanism of tumor suppressor genes. This genetic-molecular pathway for cancer may separate a subset of patients with different prognoses and eventually different responses to specific therapies. AIM: To analyze the methylation pattern of important genes related to different carcinogenic mechanisms in patients with gastric cancer (GC) and the relationship with its morphological features and biological behavior. MATERIAL AND METHODS: Forty-seven fresh-frozen GC samples were selected. The methylation-specific PCR (MSP) test was used to analyze promoter methylation status for genes MLH1, CDKN2A (p16), APC, CDH1 (Cadherin E) and FHIT. Follow-up and complete morphological features were obtained for all cases. RESULTS: We found methylation in at least one of the genes studied in 83% of the cases. The frequencies of promoter hypermethylation of MLH1, CDKN2A, APC, CDH1 and FHIT were 31%, 43%, 46%, 80% y 62%, respectively. We found a relationship between APC methylation and good histological differentiation (p =0.03); CDH1 methylation with diffuse type by Lauren and 3 or more metastasic lymph nodes (p <0.05); FHIT, CDKN2A and CDH1 methylation and female condition (p <0.04). We also found a non-significant relationship between CDKN2A methylation and better survival (p =0.07). CONCLUSIONS: The high frequency promoter methylation found confirms its importance in gastric carcinogenesis. The finding of alterations in the methylation pattern of genes studied and its association with prognostic factors is a helpful tool in the search for new criteria in clinical and therapeutic decision making.

[Frequency of K-ras mutation in biliary and pancreatic tumors]. / Mutación del gen K-ras en tumores pancreático-biliares.

BACKGROUND: The ras gene family (H-ras, N-ras and K-ras) are oncogenes that mutate frequently in human cancer, specially in tumors of the biliary tract and pancreas. AIM: To determine the frequency of K-ras gene codon 12 mutation in pancreatic and biliary tumors. MATERIAL AND METHODS: Samples of 35 gallbladder, 15 ampulla of Vater, 10 biliary tract and 9 pancreatic tumors, were analyzed. The tumor tissue was microdissected from paraffin embedded biopsies. The mutation was detected by a combination of polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). RESULTS: Overall, 46% of samples had K-ras gene mutations. Mutation frequency was 80, 56, 50 and 29% for ampulla of Vater, pancreatic, biliary tract and gallbladder tumors, respectively. When compared with the rest, gallbladder tumors had a significantly lower frequency of the mutation. Median survival for biliary tract tumors was 6 months, compared with 65 months for gallbladder tumors (p <0.05). CONCLUSIONS: Gallbladder carcinoma had the lower frequency of K-ras mutation, when compared with pancreatic, biliary tract and ampulla of Vater tumors.

[Promoter methylation profile in breast cancer]. / Patrón de metilación génico en el cáncer de mama.

BACKGROUND: Genomic DNA methylation, mutations and allelic deletions explain the inactivation of genes involved in cell proliferation and cell cycle control mechanisms. AIM: To analyze the methylation pattern of important genes related to different carcinogenic mechanisms in patients with breast cancer and the relationship with its biological behavior. MATERIAL AND METHODS: Seventy fresh-frozen breast cancer samples were selected. The methylation specific PCR (MSP) test was used to analyze promoter methylation status for genes CDKN2A (p16), hMLH1, APC, CDH1 (Cadherin E) and FHIT. RESULTS: We found methylation in at least one of the genes studied in 88% of cases and in 3 or more genes in 40.5% of cases. The frequencies of promoter hypermethylation of CDKN2A, hMLH1, APC, CDH1 and FHT were 41.4%, 11.4%, 52.9%, 70% and 42.9%, respectively. We found a relationship between CDKN2A methylatlon and better survival (p=0.002). CDH1 methylation and poor histological differentiation (p=0.007), hMLH1 methylation and non-Mapuche ethnicity (p=-0.03), APC methylation and larger tumor size (p<0.05), FHIT methylatton and lack of estrogen rectptor IHC expression (p<0.05). CONCLUSIONS: The high frequency of promoter methylation in patients with breast cancer confirms its role in breast carcinogenesis. The finding of alterations in the methylation pattern of genes studied and its association with prognostic factors is a helpful tool in the search of new criteria for clinical and therapeutic decision making.

[Association between Helicobacter pylori genotype and the severity of gastritis in infected adults]. / Relación de la genotipificación de Helicobacter pylori con la forma e intensidad de la gastritis en población adulta portadora de patología gástrica benigna.

BACKGROUND: The damaging capacity of Helicobacter pylori is variable and depends, in part, on its genetic polymorphism. AIM: To study H pylori genes vacA, cagA and iceA and the relationship of these genotypes with the features of acute damage in chronic gastritis. MATERIAL AND METHODS: Gastric endoscopic biopsies were obtained in 75 adults for pathological study and genetic typification of H pylori by specific PCR. RESULTS: In only 64 cases, complete information was available. In 53 of these, there was H pylori infection demonstrated by PCR. Twenty one percent had infection by two or more H pylori strains, vacA gene had genotypes s2/m2, s1/m1 and s1/m2 in 36, 25 and 8% of cases respectively, cagA gene was present in 49% of infected patients. iceA gene had genotypes iceA 1 ad iceA 2 in 15 and 60% of patients respectively. The presence of cagA or alleles s1/m1 and s1/m2 of vacA gene was directly correlated with polymorphonuclear infiltration and the severity of epithelial damage. The genotype s2/m2 of vacA gene was significantly associated with a milder or absent mucosal damage. No association was found between iceA alleles and the pathological features of gastritis. CONCLUSIONS: Alleles of vacA and cagA genes of H pylori are associated with the severity of gastric mucosal damage.