High-resolution Melting Profiles Research Articles

New genetic markers for 100% arabica coffee demonstrate high discriminatory potential for InDel-HRM-based coffee authentication

Food authenticity is crucial in today's society, given the heightened consumer awareness and attention to the products they consume. Reliable and efficient techniques are needed to quickly detect potential food adulterations that can negatively impact product quality and economic value. Coffee, a globally traded agricultural product, holds immense economic importance, with an estimated value of USD 83 billion. It is widely consumed and recognized as a functional food that provides minerals (K, Mg, Mn, Cr), niacin, and antioxidants. However, the preferred coffee species, Coffea arabica, known for its superior drink quality, is often adulterated with Coffea canephora (Robusta and Conilon) beans, even in 100% Arabica coffee. To distinguish between these two coffee species, a comprehensive study was conducted using a robust approach to identify differences in Single-Ortholog Copy (SOC) based on InDel regions in these gene pairs. These differences were validated using a meticulous methodology that considered variations in amplicon size: electrophoretic profile, and high-resolution melting (HRM). The innovative combination of InDels and HRM resulted in highly distinctive HRM profiles, outperforming SNP-based methods previously used. The targeted InDel approach utilized in this study facilitated precise quantification of Coffea species beans with a detection sensitivity of 0.5%. The study's findings establish the reliability and accuracy in distinguishing between the two coffee species, showcasing the valuable application of InDels for quality control and ensuring the authenticity of coffee beans. This pioneering research contributes to the advancement of authenticity verification methods for both imported and exported coffee beans, as well as in future studies that require significant genetic differences between these species, such as C. arabica and C. canephora.

Consider Non-Syndromic Mitochondrial Disorders as Part of the Pediatric Phenotypic Spectrum

With interest we read the article by Ardissone et al. about a retrospective, singlecenter study of 150 pediatric patients with a genetically confirmed mitochondrial disorder (MID) due to a pathogenic mtDNA variant (mtDNA point mutation or single mtDNA deletion) [1]. The study is excellent but has limitations that are cause of concerns and should be discussed. A limitation of the study is that only syndromic MIDs were included [1]. Since MIDs often do not fit into any of the syndromic phenotypes, we should know whether these patients were deliberately excluded or did not appear at the study center at all. We should know whether the entire group of 902 patients also included patients with nonsyndromic MIDs. Another limitation is that heteroplasmy rates were only determined by estimating the densitometric ratio between wild-type and mutant mtDNA [1]. Heteroplasmy is usually determined by a number of quantitative methods, such as Sanger sequencing, high performance liquid chromatography, pyrosequencing, Snapshot, high resolution melt profiling (HRM), temporal temperature gradient gel electrophoresis (TTGE), invader assay, amplification refractory mutation system (ARMS), endonuclease method using Surveyor nuclease, and next generation sequencing (NGS) [2]. These techniques usually provide more accurate results than semi-quantitative estimation. There is a discrepancy between the statement in the method section ("only few data were not available from all patients”) and the results section (“CSF data were available from only 17 %, follow-up data only from 47%, and biochemical data only from 58 % of patients”) [1]. This discrepancy should be clarified. Another limitation is that the total observation time up to the last follow-up was not given [1]. Knowing this latency is important to assess the speed of disease progression and estimate the outcome. There is also a discrepancy between the statement in the result section that “outcome data were evaluated from 70 patients” and the detailed description in the results section, which included 71 patients. Which number is correct? Surprisingly, the biochemical examination was normal in almost a quarter of the patients with Leigh syndrome. We should know if these results are from studies of just muscle or fibroblasts, or if other tissues have been studied as well If only muscle or fibroblasts were examined, we should know whether or not these tissues were clinically affected in the one quarter of the Leigh patients. Surprisingly, only two thirds of the MELAS patients presented with stroke-like episodes (SLEs), which are pathognomonic for MELAS. We should know if this was due to the short observation or follow-up time in these patients. According to what criteria were these patients without SLE classified as MELAS? Were the Hirano or Japanese criteria applied? We should also know how “MELAS-like” was defined. There is no mention of the underlying mutations in the PEO patients. It would be interesting to know if these patients all carried a single mtDNA deletion, a nuclear DNA-located variant, or an mtDNA point mutation. Overall, the interesting study has limitations that call the results and their interpretation into question. With a prospective design and extensive and long-term follow-up, the study goals could be better achieved.

Analysis of Salivary Bacterial Community by Direct PCR and High Resolution Melting Curve and Its Forensic Applications.

To develop a rapid test for salivary bacterial community based on direct PCR (dPCR) and high resolution melting (HRM) curve analysis, to evaluate its application value in forensic medicine. The salivary bacteria were collected by centrifugation and then resuspended in Tris-EDTA (TE) buffer, and directly used as the template for amplification and HRM curve analysis (dPCR-HRM) of the 16S rDNA V4 region. The genotype confidence percentage (GCP) of the HRM profiles compared with the reference profile was calculated. The template DNA was extracted by traditional kit and then PCR-HRM (namely kPCR-HRM) was used as reference to validate the feasibility of dPCR-HRM. The gradient dilution templates, population samples and simulated salivary stains were analyzed by dPCR-HRM to evaluate its sensitivity, typing ability and adaptability. Using dPCR-HRM method, the HRM profiles of salivary bacterial community were obtained within 90 minutes. The GCP between dPCR-HRM and kPCR-HRM was greater than 95.85%. For general individuals, the HRM type of bacterial community could be determined with 0.29 nL saliva by dPCR-HRM. The 61 saliva samples could be divided into 10 types. The typing of salivary stains deposited within 8 h was the same as those of fresh saliva (GCP>90.83%). dPCR-HRM technology can be used for rapid typing of salivary bacterial community, and has the advantage of low cost and simple operation.

SSR-HRM molecular characterization of the Colombian cultivated germplasm of Capsicum chinense Jacq. (Solanaceae)

This study was the first evaluation of the genetic diversity of cultivated populations of habanero pepper (Capsicum chinense) in Colombia using SSR-HRM. Three habanero pepper lines were characterized with eight microsatellite markers using the High-Resolution Melt (HRM) technique. Twenty-seven individuals from the HL-original line and 30 individuals each from derived lines HL-70 and HL67 were genotyped. Three microsatellites were monomorphic, and five were polymorphic; however, a high allelic diversity was detected in the homozygous state in the 87 individuals. The Ng8 marker differentiated the HL-original and HL-67 lines from the HL-70 line with HRM profiles. The analysis of molecular variance (AMOVA) revelated that 52% of the genetic variation existed within lines. The HL-67 line was more similar to the HL-original line than to the HL-70 line. HL-70 recorded the highest genetic diversity for the derived lines and, therefore, could be used in a new breeding program. In contrast, the HL-67 line, because of its high genetic homogeneity, could potentially be used to evaluate different environmental conditions to find optimal conditions that increase productivity and pungency. Finally, comparing the HRM profiles with the monomorphic markers (Ng 33, Ng 18 and Ng 10) differentiated the C. chinense and C. frutescens species, which was difficult because of high morphologic similarity between these two species and is usually evaluated at the flowering stage, while HRM profiles can be done at any plant stage.

Rapid screening of UPB1 gene variations by high resolution melting curve analysis.

The present study aimed to analyze gene mutations in patients with β-ureidopropinoase deficiency and establish a rapid detection method for β-ureidopropinoase (UPB1) pathogenic variations by high resolution melting (HRM) analysis. DNA samples with known UPB1 mutations in three patients with β-ureidopropinoase deficiency were utilized to establish a rapid detection method for UPB1 pathogenic variations by HRM analysis. Further rapid screening was performed on two patients diagnosed with β-ureidopropinoase deficiency and 50 healthy control individuals. The results showed that all known UPB1 gene mutations can be analyzed by a specially designed HRM assay. Each mutation has specific HRM profiles which could be used in rapid screening. The HRM method could correctly identify all genetic mutations in two children with β-ureidopropinoase deficiency. In addition, the HRM assay also recognized four unknown mutations. To conclude, the results support future studies of applying HRM analysis as a diagnostic approach for β-ureidopropinoase deficiency and a rapid screening method for UPB1 mutation carriers.

QTL mapping for long juvenile trait in soybean accession AGS 25 identifies association between a functional allele of FT2a and delayed flowering

Soybean is a quantitative photoperiod sensitive short-day crop. In Asian countries, soybean is grown under rain-fed conditions. However, late planting due to delayed onset of the rainy season results in the reduced biomass leading to considerable yield losses. Long juvenility (LJ) trait in soybean delays flowering and helps in gaining biomass under short days. Our objective of this work was to understand the molecular basis of long juvenility in a genetic resource AGS 25. In this study, we used recombinant inbred lines (RILs) developed from an LJ genotype AGS 25 and conventional juvenile (CJ) genotype JS 93–05 and mapped a major quantitative trait locus (QTL) explaining upto 39.7% of the phenotypic variation. The identified QTL carried two candidate flowering genes FT2a and FT2b, in which genomic DNA sequencing of coding DNA sequence identified the former with a functional SNP variation but the latter was found monomorphic. The identified mutation in FT2a could be associated with LJ and validated in another segregating population (F2 of SL 958 × AGS 25). The novel mutation caused a non-synonymous substitution of conserved glycine with aspartic acid (G169D) and the mechanism of LJ through this mutation is to be further divulged. This identified mis-sense mutation found associated with delayed flowering in two previous reports. The identified new functional SNP created differential HinfI (e9-CAPS) restriction sites and high-resolution melt (HRM) profiles (e9-HRM) for CJ and LJ parents and are associated with delayed flowering in RILs and F2 population (SL 958 × AGS 25).

Salmonella Contamination of Broiler Chicken Carcasses at Critical Steps of the Slaughter Process and in the Environment of Two Slaughter Plants: Prevalence, Genetic Profiles, and Association with the Final Carcass Status

Salmonella is a foodborne pathogen commonly associated with poultry products. The aims of this work were to (i) estimate the impact of critical steps of the slaughter process on Salmonella detection from broiler chicken carcasses in two commercial poultry slaughter plants in Quebec, Canada; (ii) investigate the presence of Salmonella in the slaughter plant environment; (iii) describe, using a high-resolution melting (HRM) approach, the HRM Salmonella profiles and serotypes present on carcasses and in the slaughter plant environment; and (iv) evaluate whether the HRM flock status after chilling could be predicted by the flock status at previous steps of the slaughter process, the status of previous flocks, or the status of the processing environment, for the same HRM profile. Eight visits were conducted in each slaughter plant over a 6-month period. In total, 379 carcass rinsates from 79 flocks were collected at five critical steps of the slaughter process. Environmental samples were also collected from seven critical sites in each slaughter plant. The bleeding step was the most contaminated, with >92% positive carcasses. A decrease of the contamination along the slaughtering process was noted, with carcasses sampled after dry-air chilling showing ≤2.5% Salmonella prevalence. The most frequently isolated serotypes were Salmonella Heidelberg, Kentucky, and Schwarzengrund. The detection of the Salmonella Heidelberg 1-1-1 HRM profile on carcasses after chilling was significantly associated with its detection at previous steps of the slaughter process and in previously slaughtered flocks from other farms during a same sampling day. Results highlight the importance of the chilling step in the control of Salmonella on broiler chicken carcasses and the need to further describe and compare the competitive advantage of Salmonella serotypes to survive processing. The current study also illustrates the usefulness of HRM typing in investigating Salmonella contamination along the slaughter process.

Полимеразная цепная реакция в реальном времени как метод быстрого генотипирования пациенток с миомой матки на примере трех генетических полиморфизмов

Study Objective: To assess the efficacy of methods for DNA gene typing (direct sequencing and two variants of real-time PCR) for routine analysis of large groups; to compare the prevalence of genotypes of three single nucleotide polymorphisms in groups of women with uterine leiomyoma (with a separate analysis of a group with a family history of the disease) and in controls. Study Design: comparative study. Materials and Methods. Subjects were divided into groups using the case-control principle. DNA gene typing results for two groups of patients were analysed. Also, we analysed prevalence of genotypes in study group (patients with uterine leiomyoma) and in controls (patients without a history of uterine leiomyoma and a family history of the disease). The primary method used for genotyping was direct sequencing with genotype imaging. On the second stage, we tested two different PCR-based genotyping methods. Study Results. Patients with uterine leiomyoma were subject to genotyping using three single nucleotide polymorphisms (rs3020434, rs124577644, rs12637801) in ESR1, FBN2, and KCWMB2 introns. We have identified significant differences in prevalence of genotypes between the study group and controls. Polymorphism prevalence is statistically different between patients with leiomyomas, controls and women with a family history of the disease. It is demonstrated that the use of the two variants of real-time PCR testing instead of direct sequencing speeds up results; these methods are a less expensive and less labour-intensive tool for genotyping of single nucleotide polymorphisms in large groups. Conclusion. Real-time PCR testing can be used for express and efficient analysis of single nucleotide polymorphisms associated with uterine leiomyoma. Both methods (PCR genotyping with the use of oligonucleotide probes and analysis of high-resolution melting profiles) tested in this paper make it possible to get unambiguous results in 97–99% of samples. Keywords: uterine leiomyoma, single nucleotide polymorphisms, family proneness.

CRISPR 2 PCR and high resolution melting profiling for identification and characterization of clinically-relevant Salmonella enterica subsp. enterica

BackgroundNontyphoidal Salmonella spp. constitute a major bacterial cause of food poisoning. Each Salmonella serotype causes distinct virulence to humans.MethodA small cohort study was conducted to characterize several aspects of Salmonella isolates obtained from stool of diarrheal patients (n = 26) admitted to Phayao Ram Hospital, Phayao province, Thailand. A simple CRISPR 2 molecular analysis was developed to rapidly type Salmonella isolates employing both uniplex and high resolution melting (HRM) curve analysis.ResultsCRISPR 2 monoplex PCR generated a single Salmonella serotype-specific amplicon, showing S. 4,[5],12:i:- with highest frequency (42%), S. Enteritidis (15%) and S. Stanley (11%); S. Typhimurium was not detected. CRISPR 2 HRM-PCR allowed further classification of S. 4,[5],12:i:- isolates based on their specific CRISPR 2 signature sequences. The highest prevalence of Salmonella infection was during the summer season (April to August). Additional studies were conducted using standard multiplex HRM-PCR typing, which confirmed CRISPR 2 PCR results and, using a machine-learning algorithm, clustered the majority of Salmonella serotypes into six clades; repetitive element-based (ERIC) PCR, which clustered the serotypes into three clades only; antibiogram profiling, which revealed the majority resistant to ampicillin (69%); and test for extended spectrum β-lactamase production (two isolates) and PCR-based detection of bla alleles.ConclusionCRISPR 2 PCR provided a simple assay for detection and identification of clinically-relevant Salmonella serotypes. In conjunction with antibiogram profiling and rapid assay for β-lactamase producers, this approach should facilitate detection and appropriate treatment of Salmonellosis in a local hospital setting. In addition, CRISPR 2 HRM-PCR profiling enabled clustering of S. 4,[5],12:i:-isolates according to CRISPR 2 locus signature sequences, indicative of their different evolutionary trajectories, thereby providing a powerful tool for future epidemiological studies of virulent Salmonella serotypes.

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.

HRM and 16S rRNA gene sequencing reveal the cultivable microbiota of the European sea bass during ice storage

The total cultivable microbiota of the ice-stored European sea bass (Dicentrarchus labrax), the most important commercial fish species of the Mediterranean aquaculture, was determined using 16S rRNA gene sequence analysis. High Resolution Melting (HRM) curve profiles and sequencing analysis (V3–V4 region of the 16S rRNA gene) were used respectively for the differentiation and identification of the collected isolates from six time intervals (day 0, 4, 8, 12, 14 and 16) while fish were stored in ice. HRM analysis differentiated the unknown microbiota in ten groups (208 isolates) and in two single isolates based on their HRM curve profiles. The isolates with HRM profiles which were >91% similar within each group were found to belong to the same species using sequencing analysis. Thus, the ten groups consist of representatives of Psychrobacter glacincola, Ps. alimentarius, Ps. cryohalolentis, Ps. maritimus, Ps. fozii, Pseudomonas sp., Paeniglutamicibacter sp., Carnobacterium sp., Leucobacter aridicolis and Bacillus thuringiensis. Based on this approach, Ps. cryohalolentis was found to be the most dominant phylotype at the beginning of fish shelf-life compared to other species. The abundance of this bacterium decreased throughout storage, while Ps. glacincola increased and dominated at the time of the sensory minimum acceptability (day 14) and rejection (day 16). To conclude, HRM could be used for the rapid determination of sea bass microbiota, using the representatives of each group as reference bacterial strains, in order for scientists to solve rapidly stakeholders problems related with microbial quality or safety of fish.

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.

Three-gene PCR and high-resolution melting analysis for differentiating vertebrate species mitochondrial DNA for biodiversity research and complementing forensic surveillance

Reliable molecular identification of vertebrate species from morphologically unidentifiable tissue is critical for the prosecution of illegally-traded wildlife products, conservation-based biodiversity research, and identification of blood-meal hosts of hematophagous invertebrates. However, forensic identification of vertebrate tissue relies on sequencing of the mitochondrial cytochrome oxidase I (COI) ‘barcode’ gene, which remains costly for purposes of screening large numbers of unknown samples during routine surveillance. Here, we adapted a rapid, low-cost approach to differentiate 10 domestic and 24 wildlife species that are common in the East African illegal wildlife products trade based on their unique high-resolution melting profiles from COI, cytochrome b, and 16S ribosomal RNA gene PCR products. Using the approach, we identified (i) giraffe among covertly sampled meat from Kenyan butcheries, and (ii) forest elephant mitochondrial sequences among savannah elephant reference samples. This approach is being adopted for high-throughput pre-screening of potential bushmeat samples in East African forensic science pipelines.

Nanoarray Digital Polymerase Chain Reaction with High-Resolution Melt for Enabling Broad Bacteria Identification and Pheno-Molecular Antimicrobial Susceptibility Test.

Toward combating infectious diseases caused by pathogenic bacteria, there remains an unmet need for diagnostic tools that can broadly identify the causative bacteria and determine their antimicrobial susceptibilities from complex and even polymicrobial samples in a timely manner. To address this need, a microfluidic and machine-learning-based platform that performs broad bacteria identification (ID) and rapid yet reliable antimicrobial susceptibility testing (AST) is developed. Specifically, this platform builds on "pheno-molecular AST", a strategy that transforms nucleic acid amplification tests (NAATs) into phenotypic AST through quantitative detection of bacterial genomic replication, and utilizes digital polymerase chain reaction (PCR) and digital high-resolution melt (HRM) to quantify and identify bacterial DNA molecules. Bacterial species are identified using integrated experiment-machine learning algorithm via HRM profiles. Digital DNA quantification allows for rapid growth measurement that reflects susceptibility profiles of each bacterial species within only 30 min of antibiotic exposure. As a demonstration, multiple bacterial species and their susceptibility profiles in a spiked-in polymicrobial urine specimen were correctly identified with a total turnaround time of ∼4 h. With further development and clinical validation, this platform holds the potential for improving clinical diagnostics and enabling targeted antibiotic treatments.

Characterization of Listeria monocytogenes serovar 1/2a, 1/2b, 1/2c and 4b by high resolution melting analysis for epidemiological investigations

This study aimed to characterize serovar 1/2a, 1/2b, 1/2c and 4b of Listeria monocytogenes cultures based on High Resolution Melting (HRM) profiles, targeting 53 fragments in the region comprising prs, Listeria Pathogenicity Island-1 (LIPI-1) and ldh loci, and 28 fragments of inlAB operon. Fifty L. monocytogenes cultures (28 of lineage I, 22 of lineage II) were tested. Real time PCR and EvaGreen-based HRM assays were performed, and the HRM profile for each amplicon was compared to that of L. monocytogenes EGD-e strain. Considering all fragments tested, 45 HRM profiles were identified (Diversity Index = 99.35). Similarity analysis identified two main clusters: the first consisted of 25 cultures, including all 1/2a and 1/2c strains, except for three isolates from food of serovar 4b; the second group only included 1/2b and 4b isolates. Eighteen out of targeted amplicons showed constant HRM characteristics irrespective of the serovar/lineage, and hlyA displayed the most stable melting behavior. Conversely, thirteen amplicons were specific for 1/2b and 4b isolates, showing major variations within actA, followed by prs, prfA, mpl and plcB genes. A fragment targeting an intragenic region (part of plcA and part of an unknown gene) had a melting profile allowing differentiation between 4b and 1/2b isolates showing different Tm variants. Amplicons of inlA and inlB exhibited the largest intra-strain melting differences, and one inlB fragment could allow discriminating between 4b and 1/2b cultures, as well as between lineages. A greater level of genetic diversity amongst 1/2a cultures compared to 1/2c, 1/2b and 4b isolates was observed, with variations identified in LIPI-1, as well as within inlA and inlB genes. Sequencing analysis of amplicons with differential HRM profile from EGD-e confirmed point mutations. The study findings underlines that HRM-based approach may be useful for bacterial subtyping for epidemiological purposes when sequencing-based methods are not available.

High-resolution melting of multiple barcode amplicons for plant species authentication

In recent years, species identification in herbs has attracted considerable attention due to several cases of fraud; hence inexpensive high-throughput authentication methods are highly welcomed. Species authentication is often performed through DNA analysis and several specific regions (barcodes) are considered suitable. Each barcode (Bar) possesses different qualities in terms of universality and discrimination power. A multiplexed format where information can be extracted simultaneously from several barcode regions is seemingly appropriate to ensure the power of both universality and discrimination. In this approach, we amplified DNA from five different barcode regions in a multiplexed PCR format followed by high-resolution melting (HRM). This multiplexed Bar-HRM approach was first applied to plants spanning the plant kingdom and then gradually narrowing down the genetic variability within the Lamiaceae and the Solanaceae families to finally reach closely related cultivars. Universality was demonstrated through distinct melting profiles obtained for species originating from 29 different families spanning the angiosperms, gymnosperm, mosses, and liverwort (Marchantiophyta). Discrimination power was retained for species, sub-species, and a few cultivars through the application of multivariate statistics to the high-resolution melting profiles. This preliminary investigation has shown the potential to discriminate a vast amount of species within the whole plant kingdom. It requires no a priori knowledge of the species' DNA sequence and occurs in a closed system within 2.5 h at a reduced cost per sample compared to other DNA based approaches.

Improved molecular typing of toxigenic Clostridium difficile strains affecting animal and human health

Clostridium difficile is a Gram-positive, spore forming bacterium, which remains a formidable pathogen as the etiological agent of C. difficile infection (CDI). Substantial effort goes into diagnosis of CDI and characterisation of circulating toxigenic C. difficile strains for epidemiology and infection prevention and control. Currently, molecular typing of C. difficile requires 9 days following diagnosis through PCR ribotyping and multilocus variable number tandem repeat (VNTR) analysis. There is a need for more rapid typing methods to investigate possible linkage between CDI cases in healthcare settings. This study developed a one-step, closed tube real-time PCR and high resolution melt (HRM) assay targeting the intergenic spacer region (ISR) and several VNTR loci, with results generated in 2.5 h. The discriminatory power of the PCR-HRM assay was investigated by typing previously characterised toxigenic clinical and animal C. difficile isolates (n=90). Through comparison of HRM profiles targeting the ISR of isolates belonging to 17 PCR ribotypes, 13 HRM genotypes were recognised with 11 PCR ribotypes resolved from each other. Using correlation between HRM data and known VNTR repeat numbers at the B7, C6 and G8 loci, VNTR repeat numbers for isolates could be predicted within an average absolute difference of 1.8 at the B7 locus, 2.1 at the C6 locus, and 2.5 at the G8 locus. These results suggest that a PCR-HRM assay with a multilocus panel targeting ISR and selected VNTR loci could form part of an improved molecular typing scheme for toxigenic C. difficile strains that is faster than currently available methods.

High resolution melting analysis for the characterization of lineage II Listeria monocytogenes serovars 1/2a and 1/2c based on single nucleotide polymorphisms identification within the Listeria Pathogenicity Island-1 and inlAB operon: a novel approach for epidemiological surveillance.

A high resolution melting (HRM) assay was developed for characterizing lineage II Listeria monocytogenes based on the amplification and the melting profiles analysis of 81 fragments targeting the region from the prs to ldh loci, including the Listeria Pathogenicity Island-1 (LIPI-1) genes and the inlAB operon. Real-time PCR and HRM protocols were standardized using 10 replicate assays from L. monocytogenes EGD-e reference strain (serovar 1/2a). Twenty wild-type isolates of serovar 1/2a and two of serovar 1/2c were tested, and differences between EGD-e strain and the wild-type isolates were defined if the melting temperature (Tm ) of an amplicon was not within the lower and the upper limits calculated from replicate testing on EGD-e. The analysis revealed 17 and 19 HRM profiles with respect to prs/LIPI-1/ldh and inlAB target regions (Simpson's Index of Diversity 0·979 and 0·983) respectively. The 1/2c cultures showed 98·1% similarity to melting characteristics with EGD-e, whilst 1/2a isolates had the greatest heterogeneity that was related to inlA, inlB and actA genes. Sequencing of amplicons generating different Tm values from EGD-e confirmed the presence of point mutations. This method was useful for L. monocytogenes subtyping based on single nucleotide polymorphisms detection through the melting behaviour analysis of main virulence genes. The study underlines the effectiveness of HRM in differentiating L. monocytogenes strains with high discriminatory power, thus rendering it useful for epidemiological surveillance.

Microsatellite High-Resolution Melting (SSR-HRM) to Track Olive Genotypes: From Field to Olive Oil.

The need to support food labelling has driven to the development of PCR-based techniques suitable for food analysis. DNA-based markers have been successfully employed for varietal tracing in Protected Designation of Origin (PDO) olive oils. In this study, we report a fast, sensitive, and easy-to-use strategy for PDO olive varietal identification. To achieve this aim four different DNA extraction methods were tested and compared, based on initial volume, extraction time, the gDNA concentration, and quality ratios. The optimized DNA extraction protocol from extra virgin olive oils, based on CTAB-hexane-chloroform extraction, proved to be the most effective. High-resolution melting (HRM) DNA assay was developed based on nuclear microsatellites (gSSR) and plastid DNA (cpDNA) aiming an accurate identification of the olive varieties present in the olive oil samples. After PCR reproducibility evaluation, six molecular markers: three SSRs and three cpDNA loci were chosen based on their discrimination power. The SSR-HRM strategy assays were designed to target UDO99-011, UDO99-039, UDO99-024, and ssrOeUA-DCA16 loci. All SSR-PCR products generated from these primers were analyzed by capillary electrophoresis (CE) for HRM data validation. The SSR coupled with HRM melting curve analysis generated 14 HRM profiles sufficient to genotype all varieties, highlighting their potential use for varietal discrimination. The locus ssrOeUA-DCA16 generated a specific melting curve that allow a high-throughput discrimination of the Picual and Cobrançosa varieties in olive oil samples. Further, the UDO99-024 was also tested by SSR-HRM assay in commercial olive oil samples with promising results. Considering time, cost, and performance SSR-HRM proved to be a reliable method suitable for varietal tracing of olive oils. PRACTICAL APPLICATION: Olive oil authenticity is a form of protecting producers and consumers against fraudulent practices. Herein, we present a DNA barcode suitable for the identification of olive varieties, allowing an accurate identification of the olive varieties in olive oil samples using SSR-HRM assay. Its applicability in commercial olive oil samples is viable. This methodology can be used as a tool for Extra Virgin Olive Oil (EVOO) adulterations detection.

The potential of aerosol eDNA sampling for the characterisation of commercial seed lots.

Seed shipments, silos and storage houses often contain weed seeds or seeds of restricted crops such as undeclared genetically modified (GM) varieties. Random sub-sampling is the favoured approach to detect unwanted biological materials in seed lots but is prohibitively expensive or else ineffective for the huge volumes of seeds moved in commercial operations. This study uses maize and cowpea seed admixtures as an exemplar to evaluate the feasibility of using aerosol sampling of “seed dust” as an alternative to seed sub-sampling. In an initial calibration phase, qPCR of the rbcL barcode followed by high-resolution melting (HRM) of a DNA titration series revealed a strong linear relationship between mix composition and HRM profiles. However, the relationship became skewed when flour mixes were used to build the titration, implying a DNA extraction bias favouring cowpea. Aerosol samples of seed dust above a titration of mixed seed samples were then collected along vertical and lateral axes. Aerosols were characterised by light microscopy, qPCR-HRM and next-generation DNA sequencing (Illumina MiSeq). Both molecular approaches again showed bias but this time in a reverse direction to flour samples. Microscopic examination of the aerosol sample suggested this divergence could be attributed to differences in abundance of airborne starch particles. Despite the bias, it was nevertheless possible to estimate relative abundance of each species using the abundance of minibarcodes. In light of these results we explore the feasibility of aerosol sampling for commercial seed lot characterisation.