A circulating cell-free DNA methylation signature for the detection of hepatocellular carcinoma.

To address the shortcomings of current hepatocellular carcinoma (HCC) surveillance tests, we set out to find HCC-specific methylation markers and develop a highly sensitive polymerase chain reaction (PCR)-based method to detect them in circulating cell-free DNA (cfDNA). The analysis of large methylome data revealed that Ring Finger Protein 135 (RNF135) and Lactate Dehydrogenase B (LDHB) are universally applicable HCC methylation markers with no discernible methylation level detected in any other tissue types. These markers were used to develop Methylation Sensitive High-Resolution Analysis (MS-HRM), and their diagnostic accuracy was tested using cfDNA from healthy, at-risk, and HCC patients. The combined MS-HRM RNF135 and LDHB analysis detected 57% of HCC, outperforming the alpha-fetoprotein (AFP) test's sensitivity of 45% at comparable specificity. Furthermore, when used with the AFP test, the methylation assay can detect 70% of HCC. Our findings suggest that the cfDNA methylation assay could be used for HCC liquid biopsy.

High-throughput identification of tuna (Thunnus spp.) larvae in the Gulf of Mexico using unlabelled-probe high-resolution melting analysis.

The genus Thunnus (family Scombridae) comprises eight species of tunas of which all but one are targeted by industrialized fisheries. Although intact individuals of these species can be distinguished by morphological characteristics, researchers and managers often rely on dressed, frozen, juvenile or larval fish samples, which often necessitates the identification of molecular species. Here the authors investigate short amplicon (SA) and unlabelled probe high-resolution melting analysis (UP-HRMA) as a low-cost, high-throughput molecular genotyping assay capable of distinguishing between albacore tuna (Thunnus alalunga), blackfin tuna (Thunnus atlanticus), bigeye tuna (Thunnus obesus), Atlantic bluefin tuna (Thunnus thynnus) and yellowfin tuna (Thunnus albacares) in the Gulf of Mexico. Although SA-HRMA of variable regions in the NADH dehydrogenase subunit 4 (ND4) and subunit 5 (ND5), and subunit 6 (ND6) of the mtDNA genome did yield some species-specific diagnostic melting curves (e.g., ND4 assay can reliably distinguish Atlantic bluefin tuna), genotype masking produced excessive variation in melting curves for reliable multi-species identification. To minimize the genotyping masking of SA-HRMA a 26 base pair long UP containing four SNPs was developed within a 133 bp segment of ND4. The UP-HRMA is able to reliably distinguish Gulf of Mexico species T. thynnus, T. obesus, T. albacares and T. atlanticus by UP melting temperature at 67, 62, 59 and 57°C, respectively. The developed UP-HRMA assay is a lower-cost, higher-throughput, alternative to previously published molecular assays for tuna identification that can be easily automated for large data sets, including ichthyological larval surveys, fisheries specimens lacking distinguishing morphological characteristics or detection of fraudulent trading of tuna species.

Molecular identification and anti-malarial drug resistance profile of Plasmodium falciparum from patients attending Kisoro Hospital, southwestern Uganda.

BACKGROUND: The evolution of malaria infection has necessitated the development of highly sensitive diagnostic assays, as well as the use of dried blood spots (DBS) as a potential source of deoxyribonucleic acid (DNA) yield for polymerase chain reaction (PCR) assays. This study identified the different Plasmodium species in malaria-positive patients, and the anti-malarial drug resistance profile for Plasmodium falciparum using DBS samples collected from patients attending Kisoro Hospital in Kisoro district, Southwestern Uganda. METHODS: The blood samples were prospectively collected from patients diagnosed with malaria to make DBS, which were then used to extract DNA for real-time PCR and high-resolution melting (HRM) analysis. Plasmodium species were identified by comparing the control and test samples using HRM-PCR derivative curves. Plasmodium falciparum chloroquine (CQ) resistance transporter (pfcrt) and kelch13 to screen the samples for anti-malarial resistance markers. The HRM-PCR derivative curve was used to present a summary distribution of the different Plasmodium species as well as the anti-malarial drug profile. RESULTS: Of the 152 participants sampled, 98 (64.5%) were females. The average age of the participants was 34.9 years (range: 2 months-81 years). There were 134 samples that showed PCR amplification, confirming the species as Plasmodium. Plasmodium falciparum (N = 122), Plasmodium malariae (N = 6), Plasmodium ovale (N = 4), and Plasmodium vivax (N = 2) were the various Plasmodium species and their proportions. The results showed that 87 (71.3%) of the samples were sensitive strains/wild type (CVMNK), 4 (3.3%) were resistant haplotypes (SVMNT), and 31 (25.4%) were resistant haplotypes (CVIET). Kelch13 C580Y mutation was not detected. CONCLUSION: The community served by Kisoro hospital has a high Plasmodium species burden, according to this study. Plasmodium falciparum was the dominant species, and it has shown that resistance to chloroquine is decreasing in the region. Based on this, molecular identification of Plasmodium species is critical for better clinical management. Besides, DBS is an appropriate medium for DNA preservation and storage for future epidemiological studies.

Possible correlation between high circulatory levels of trimethylamine-N-oxide and 2177G>C polymorphisms of hepatic flavin containing monooxygenase 3 in Kurdish Population with non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a multifactorial disorder with complicated pathophysiology. Trimethylamine-N-oxide (TMAO) has been thought to be correlated with the pathogenesis of NAFLD. The single nucleotide polymorphisms (SNPs) of hepatic flavin-containing monooxygenase 3 (FMO3) regulate the concentration of TMAO. This case-control study investigated the plasma levels of TMAO as well as its possible correlation with the frequency of specific genotype of FMO3 (-2650C>G, -2543T>A, -2177G>C, -2589C>T, -2106G>A polymorphisms) in Kurdish patients with NAFLD. METHODS AND RESULTS: In 85 confirmed NAFLD patients and 30 healthy individuals, triglycerides (TG), total cholesterol (Chol), low-density lipoprotein (LDL), high-density lipoprotein (HDL), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities were measured. TMAO was also measured using the LC-MS/MS method. High-resolution melting analysis was applied to determine FMO3 genotypes. Plasma TMAO levels were significantly higher in patients (p = 0.030). A CC genotype with a frequency of 12.9% for SNP -2177G>C was found in Kurdish NAFLD patients. The distribution of the GC genotype was also significantly different (p = 0.017). CONCLUSIONS: The current results provide documentation for high circulatory levels of TMAO and its possible correlation with the presence of the specific genotype -2177G>C FMO3 in Kurdish NAFLD patients.

High-resolution melting analysis for detection of fusion allele of FUT2.

The secretor status of ABH antigens, determined by FUT2 polymorphisms, affects susceptibility to various infectious diseases. In addition to many SNPs responsible for the nonsecretor phenotype, five nonfunctional alleles (se) resulting from copy number variations have been reported. One of the five alleles generated by an unequal crossover between FUT2 and a pseudogene (SEC1), is sefus . This allele may be misidentified as a functional allele if only common inactivating SNPs are genotyped because it contains the 3' region of the functional FUT2. Therefore, accurate detection of sefus is desirable. For this purpose, a high-resolution melting (HRM) analysis is developed for detection of sefus in which a 284bp fragment of SEC1 and sefus but not FUT2, are amplified. This HRM analysis detected sefus reliably. Thus, an initial screening or prescreening for sefus using HRM analysis seems to be useful for association studies of FUT2.

High-resolution (mtDNA) melting analysis for simple and efficient characterization of Africanized honey bee Apis mellifera (Hymenoptera:Apidae).

Analysis of the mtDNA variation in Apis mellifera L. has allowed distinguishing subspecies and evolutionary lineages by means of different molecular methods; from RFLP, to PCR-RFLP and direct sequencing. Likewise, geometric morphometrics (GM) has been used to distinguish Africanized honey bees with a high degree of consistency with studies using molecular information. High-resolution fusion analysis (HRM) allows one to quickly identify sequence polymorphisms by comparing DNA melting curves in short amplicons generated by real-time PCR (qPCR). The objective of this work was to implement the HRM technique in the diagnosis of Africanization of colonies of A. mellifera from Argentina, using GM as a validation method. DNA was extracted from 60 A. mellifera colonies for mitotype identification. Samples were initially analyzed by HRM, through qPCRs of two regions (485 bp/385 bp) of the mitochondrial cytochrome b gene (cytb). This technique was then optimizing to amplify a smaller PCR product (207 bp) for the HRM diagnosis for the Africanization of colonies. Of the 60 colony samples analyzed, 41 were classified as colonies of European origin whereas 19 revealed African origin. All the samples classified by HRM were correctly validated by GM, demonstrating that this technique could be implemented for a rapid identification of African mitotypes in Apis mellifera samples.

Duplex high resolution melting analysis (dHRMA) to detect two hot spot CYP24A1 pathogenic variants (PVs) associated to idiopathic infantile hypercalcemia (IIH).

Pathogenic variants (PVs) in CYP24A1 gene are associated with Idiopathic Infantile Hypercalcemia disease (IIH). The identification of CYP24A1 PVs can be a useful tool for the improvement of target therapeutic strategies. Aim of this study is to set up a rapid and inexpensive High Resolution Melting Analysis (HRMA)-based method for the simultaneous genotyping of two hot spot PVs in CYP24A1 gene, involved in IIH. A duplex-HRMA (dHRMA) was designed in order to detect simultaneously CYP24A1 c.428_430delAAG, p.(Glu143del) (rs777676129) and c.1186C > T, p.(Arg396Trp) (rs114368325), in peculiar cases addressed to our Laboratory. dHRMA was able to identify clearly and simultaneously both hot spot CYP24A1 PVs evaluating melting curve shape and melting temperature (Tm). This is the first dHRMA approach to rapidly screen the two most frequent CYP24A1 PVs in peculiar case, providing useful information for diagnosis and patient management in IIH disease.

Optimization and application of a high-resolution melting protocol in the characterization of avian infectious laryngotracheitis virus.

A previous sequence analysis of a US5 gene fragment of infectious laryngotracheitis virus (ILTV) performed in an Argentinian epidemiological study allowed to differentiate between wild and vaccine strains. This analysis also defined five ILTV haplotypes with specific variations at positions 461, 484, 832, 878 and 894 of the US5 gene. This characterization of viral strains may also be accomplished using the High-Resolution Melting Analysis (HRMA), which has been described as an effective, fast and sensitive method to detect mutations in PCR products. In the present study, an HRM protocol was developed with the aim of characterizing the circulating ILTV strains in Argentina. The specificity of this tool was confirmed in different DNA diluents, without interference from heterologous DNA or other cellular metabolites. Additionally, the salt concentration in the elution buffer used for DNA extraction did not alter the curve profiles. Higher concentrations of DNA (Ct≅26.0) displayed well-defined curve profiles, whereas lower concentrations (Ct≅32.5) exhibited more heterogeneous curves. The HRMA showed 97.49% concordance with the reference technique, i.e., sequencing. The HRM protocol has the capability to perform DNA amplification prior to its characterization. Thus, eventually this technique may be used simultaneously as a diagnostic tool. This advantage implies a significant reduction in the time and effort involved in sample processing.

Implementation of high-resolution melting analysis of the porcupine (PORCN) gene for molecular diagnosis of focal dermal hypoplasia: Identification of a novel mutation.

BACKGROUND: Focal dermal hypoplasia (FDH) is rare X-linked dominant disease characterized by atrophy and linear pigmentation of the skin, split hand/foot deformities and ocular anomalies. FDH is caused by mutations of the Porcupine (PORCN) gene, which encodes an enzyme that catalyzes the palmitoylation of Wnt ligands required for their secretion. High resolution melting analysis (HRM) is a technique that allows rapid, labor-efficient, low-cost detection of genomic variants. In the present study, we report the successful implementation of HRM in the molecular diagnosis of FDH. METHODS: Polymerase chain reaction and HRM assays were designed and optimized for each of the coding exons of the PORCN gene, processing genomic DNA samples form a non-affected control and a patient complying with the FDH diagnostic criteria. The causal mutation was characterized by Sanger sequencing from an amplicon showing a HRM trace suggesting heterozygous variation and was validated using an amplification-refractory mutation system (ARMS) assay. RESULTS: The melting profiles suggested the presence of a variant in the patient within exon 1. Sanger sequencing revealed a previously unknown C to T transition replacing a glutamine codon for a premature stop codon at position 28, which was validated using ARMS. CONCLUSIONS: Next-generation sequencing facilitates the molecular diagnosis of monogenic disorders; however, its cost-benefit ratio is not optimal when a single, small or medium size causal gene is already identified and the clinical diagnostic presumption is strong. Under those conditions, as it is the case for FDH, HRM represents a cost- and labor-effective approach.

Somatic mutation detection efficiency in EGFR: a comparison between high resolution melting analysis and Sanger sequencing.

BACKGROUND: High resolution melting curve analysis is a cost-effective rapid screening method for detection of somatic gene mutation. The performance characteristics of this technique has been explored previously, however, analytical parameters such as limit of detection of mutant allele fraction and total concentration of DNA, have not been addressed. The current study focuses on comparing the mutation detection efficiency of High-Resolution Melt Analysis (HRM) with Sanger Sequencing in somatic mutations of the EGFR gene in non-small cell lung cancer. METHODS: The minor allele fraction of somatic mutations was titrated against total DNA concentration using Sanger sequencing and HRM to determine the limit of detection. The mutant and wildtype allele fractions were validated by multiplex allele-specific real-time PCR. Somatic mutation detection efficiency, for exons 19 & 21 of the EGFR gene, was compared in 116 formalin fixed paraffin embedded tumor tissues, after screening 275 tumor tissues by Sanger sequencing. RESULTS: The limit of detection of minor allele fraction of exon 19 mutation was 1% with sequencing, and 0.25% with HRM, whereas for exon 21 mutation, 0.25% MAF was detected using both methods. Multiplex allele-specific real-time PCR revealed that the wildtype DNA did not impede the amplification of mutant allele in mixed DNA assays. All mutation positive samples detected by Sanger sequencing, were also detected by HRM. About 28% cases in exon 19 and 40% in exon 21, detected as mutated in HRM, were not detected by sequencing. Overall, sensitivity and specificity of HRM were found to be 100 and 67% respectively, and the negative predictive value was 100%, while positive predictive value was 80%. CONCLUSION: The comparative series study suggests that HRM is a modest initial screening test for somatic mutation detection of EGFR, which must further be confirmed by Sanger sequencing. With the modification of annealing temperature of initial PCR, the limit of detection of Sanger sequencing can be improved.

Broad diversity of simian immunodeficiency virus infecting Chlorocebus species (African green monkey) and evidence of cross-species infection in Papio anubis (olive baboon) in Kenya.

BACKGROUND: Simian immunodeficiency virus (SIV) naturally infects African non-human primates (NHPs) and poses a threat of transmission to humans through hunting and consumption of monkeys as bushmeat. This study investigated the as of yet unknown molecular diversity of SIV in free-ranging Chlorocebus species (African green monkeys-AGMs) and Papio anubis (olive baboons) within Mombasa, Kisumu and Naivasha urban centres in Kenya. METHODS: We collected blood samples from 124 AGMs and 65 olive baboons in situ, and detected SIV by high-resolution melting analysis and sequencing of PCR products. RESULTS: Simian immunodeficiency virus prevalence was 32% in AGMs and 3% in baboons. High-resolution melting (HRM) analysis demonstrated distinct melt profiles illustrating virus diversity confirmed by phylogenetic analysis. CONCLUSIONS: There is persistent evolutionary diversification of SIVagm strains in its natural host, AGMs and cross-species infection to olive baboons is occurring. Further study is required to establish pathogenesis of the diverse SIVagm variants and baboon immunological responses.

Discriminating larvae of two syntopic Cychramus species (Coleoptera, Nitidulidae) by means of bar-HRM analysis.

Molecular genetic methods are increasingly used to supplement or substitute classical morphology-based species identification. Here, we employ a COI mini-barcode coupled high-resolution melting analysis to quickly, cost-efficiently and reliably determine larvae of two closely related Cychramus (Coleoptera, Nitidulidae) species. Euclidean distance comparison (p < 0.01) and a Welch t-test of the melting point temperatures (p < 0.01) provide highly significant statistical evidence for species specific differences in melting and fluorescence curves, thus allowing the assignment of larvae to either of the two species. This protocol serves as a fast, low-cost and low-tech method to discriminate between pairs or groups of closely related species and can be adapted and applied to various ecological research questions.

Detection of HBB:c.92+5G>C and HBB:c.108delC mutations in ß-thalassemia carriers using high-resolution melting analysis.

The HBB:c.92+5G>C [known as IVSI-5 (GC)] and HBB:c.108delC [known as Cd 35 (del C)] are the ß-globin gene mutations that commonly found in Indonesia. Detection of the mutation in the ß-thalassemia carriers can be useful to prevent an increase in the number of ß-thalassemia patients. High-Resolution Melting Analysis (HRMA) is the method that can detect the mutation rapidly. The aim of this study was to detect HBB:c.92+5G>C and HBB:c.108delC mutations of the ß-thalassemia carriers using HRMA. DNA was isolated from blood archive of ten hematologically proved ß-thalassemia carriers. Detection of mutations was carried out by HRMA and then confirmed by sequencing. HRMA was performed by using two pairs of specific primers. One pair of primer targeted the region of HBB:c.92+5G>C and the other targeted the region of HBB:c.108delC. The results of detection of mutation using HRMA then were confirmed by sequencing. A specific primer pair covering the region of HBB:c.92+5G>C to HBB:c.108delC were used for sequencing. The results of HRMA showed that the HBB:c.92+5G>C and HBB:c.108delC mutations found in 50% and 30% samples, respectively. The HRMA results can be confirmed by sequencing in all samples. It can be concluded that HRMA can be used to detect HBB:c.92+5G>C heterozygote and HBB:c.108delC heterozygote mutations.

Spliceogenic analysis of BRCA1 c.439T>C (rs794727800) variant by High Resolution Melting Analysis.

Correct classification of genomic variants causing potentially aberrant splicing is of utmost importance for patient management, especially in clinically actionable genes such as BRCA1/2. In this article, we report molecular evaluation of the BRCA1 c.439T>C (rs794727800, p.Leu147=) variant based on RNA of a patient suffering with high-grade serous ovarian cancer syndrome, to add new evidence to the only in silico data available for this variant. High Resolution Melting Analysis (HRMA) was used for the first time to investigate the spliceogenicity of a BRCA1 variant. HRMA with Sanger sequencing provided evidence that the c.439C allele does not cause aberrant splicing of the BRCA1 exon 7. In addition, HRMA with Sanger highlighted a different expression of the naturally occurring BRCA1 r.442_444del (c.442_444delCAG, p.Gln148del, at DNA level) isoform between blood and tumor, in this patient. HRMA is an alternative molecular approach to analyze spliceogenic properties of the c.439T>C variant and potentially for all those BRCA1/2 variants affecting splicing sites. These new evidences allowed to classify definitively the c.439T>C variant as benign. Furthermore, the different BRCA1 r.442_444del expression opens the discussion to consider a wider classification criteria for the splicing variants, including molecular evaluation at tissue level, which is an aspect currently scarcely considered in BRCA1/2 variant classification recommendations.

High resolution melting profiles (HRMPs) obtained by magnetic induction cycler (MIC) have been used to monitor the BRCA2 status highlighted by next generation tumor sequencing (NGTS): a combined approach in a diagnostic environment.

Resistance can be the result of secondary tissue variants (STVs), which restore the open reading frame of the germline BRCA allele, producing functional BRCA protein in germline BRCA1/2 (BRCA) pathogenic variant (PV) carriers, treated with platinum-based chemotherapy or poly-(ADP-ribose) polymerase inhibitors (PARP-1). We reported recently a BRCA2 mutant high grade serous ovarian cancer (HGSOC) patient with acquired resistance to the PARP-1 olaparib due to a STV detected by next generation tumor sequencing (NGTS). The aim of this study was to evaluate the versatility of the high-resolution melting analysis (HRMA) obtained by magnetic induction cycler (MIC) to monitor the BRCA2 status in formalin-fixed paraffin-embedded (FFPE) tissue samples of this patient and to compare the results obtained by NGTS. HRMA highlighted the BRCA2 STV previously detected in the IIIrd HGSOC recurrence following the tissue BRCA2 tissue status comparing the high resolution melting profiles (HRMPs). HRMPs differentiate not only BRCA2 alleles, but also their different allele abundance. We underline that (1) the MIC uses a latest generation technology guaranteeing temperature uniformity and maintenance in each well allowing high and accurate performance to obtain reported results and (2) the HRMA maintains a high sensitivity and specificity when it is performed on FFPE samples. Finally, this study represents an additional use of the HRMA, confirming its extreme versatility in the diagnostic environment.

High Resolution Melting (HRM) Genotyping in the Genus Origanum: Molecular Identification and Discrimination for Authentication Purposes.

Origanum L. (Lamiaceae) is an important genus of medicinal and aromatic plants used since ancient times as culinary herbs and remedies in traditional medicine. Although it is a relatively small genus, intra-generic species delineation, as well as its inter-generic relationships within tribe Mentheae, are still poorly understood. High resolution melting (HRM) analysis, coupled with microsatellite markers (SSRs), could facilitate the molecular identification and characterization of certain genotypes more efficiently and relatively faster when compared to other analytical methods. In this study, 38 Origanum samples corresponding to six Origanum taxa (O. dictamnus, O. majorana, O. onites, O. scabrum, O. sipyleum, and O. vulgare subsp. hirtum) were analyzed, using six microsatellite loci. Our goal was to molecularly identify and discriminate among the selected samples and to evaluate the ability of the HRM technique as an analytical tool for the discrimination of Origanum species from Greece. The temperature-shifted melting curves produced by the HRM analysis, resulted in 98 unique HRM profiles, which enabled the discrimination of the Origanum genotypes studied. According to the similarity dendrogram based on the HRM profiles, six unique clusters were formed, each one corresponding to a single taxon. In conclusion, HRM genotyping provided a fast, cost-effective method, well suited for the molecular characterization and identification of Origanum taxa and for the authentication of the original genetic material.

BICD2 mutational analysis in hereditary spastic paraplegia and hereditary motor and sensory neuropathy.

INTRODUCTION: Mutations in the BICD2 gene are causative for an autosomal dominant form of spinal muscular atrophy (SMALED2). Further, BICD2 mutations have been implicated in hereditary spastic paraplegia (HSP), but only very few such patients have been described. In this report we aimed to investigate the frequency of BICD2 mutations in patients with HSP and hereditary motor and sensory neuropathy (HMSN) who were negative for the most common known genetic causes. METHODS: The cohorts comprised 171 HSP and 189 HMSN patients. Mutational analysis was performed with high-resolution melting analysis followed by Sanger sequencing. RESULTS: In both cohorts, we found no known or likely pathogenic mutations in the BICD2 gene. DISCUSSION: BICD2 mutations appear rather unlikely to cause a phenotype of HMSN and are a very rare cause of the HSP phenotype. Muscle Nerve 59:484-486, 2019.

High-resolution melting analysis to screen the ST18 gene functional risk variant for pemphigus vulgaris: The occasion to open a debate on its usefulness in clinical setting.

The ST18 -497-65050 T > C polymorphisms (rs17315309) exhibit a very strong association in the pathogenesis of Pemphigus Vulgaris (PV) and could represent a new potential molecular target for the treatment of disease. The present study aimed to establish a low-cost, sensitive and reliable assay using high-resolution melting curve analysis (HRMA) on magnetic induction rotor-based platform, the Magnetic Induction Cycler (MIC) (Bio molecular Systems). HRMA assay was able to identify easily and unambiguously the c.-497-65050 T > C genotypes evaluating melting curve shape and melting temperature (Tm). The results of HRMA were validated by direct DNA sequencing. The HRMA is rapid, sensitive, low-cost and high-throughput assay to screen the rs17315309 variant and could be used in clinical diagnostic laboratories.

Development and validation of a rapid method for genotyping three P-selectin gene polymorphisms based on high resolution melting analysis.

BACKGROUND: High resolution melting (HRM) analysis is one of the newer, reliable, and sensitive genotyping techniques, which offers considerable time and cost savings. P-selectin is an adhesion molecule that has a role in the initial phases of leukocyte adhesion to stimulated platelets and endothelial cells in inflammation. Multiple polymorphisms in P-selectin gene (SELP) that affect the protein sequence have been described. The aim of this study was to design, optimize, and validate a simple and rapid in-house HRM-based method for genotyping the NM_003005.3:c.992G>A (c.992G>A), NM_003005.3:c.1918G>T (c.1918G>T), and NM_003005.3:c.2266A>C (c.2266A>C) SELP polymorphisms. METHODS: Initial genotyping of three SELP polymorphisms was performed by applying polymerase chain reaction (PCR) with sequence-specific primers (SSP), which was used as a reference method for determination of analytical sensitivity. PCR-HRM was performed with primers for c.2266A>C reported in the literature. Primers for the remaining two polymorphisms were designed using Primer-BLAST. Precision testing was performed using three samples with different genotypes. For accuracy, analytical sensitivity and specificity testing, 20 wild type, 10 heterozygous, and 10 homozygous samples were chosen per polymorphism. Results were expressed as percentage of concordance with the acceptability criterion ≥95%. RESULTS: Agreement of results was 100% for all validation parameters except for analytical sensitivity for c.1918G>T and c.2266A>C, with agreement of 90%. Repeated analysis using both methods revealed an error in initial genotyping and correct genotyping by PCR-HRM, which was confirmed by Sanger sequencing. CONCLUSION: The validation confirmed PCR-HRM as a precise, accurate, and specific method for genotyping the c.992G>A, c.1918G>T, and c.2266A>C SELP polymorphisms.

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.