High-resolution Melting Analysis Research Articles

Методы генотипирования крупного рогатого скота для post-GWAS аннотирования SNPs

Abstract. Genome-wide association analysis is one of the key tools for elucidating the genetic etiology of various phenotypes, including diseases and the degree of predisposition to them. Subsequently, for statistically significant genetic markers, it is necessary to conduct validation studies on independent cohorts. These post-GWAS validation studies test genetic markers that are strongly associated with the phenotype, regardless of sample size, allowing to identify false-positive results from the initial association analysis. When choosing a genotyping technique for post-GWAS validation studies, consideration should be given to the sample size and the number of genetic markers planned to be studied, since genotyping techniques differ in throughput und cost. The aim of this paper is to describe modern methods of genotyping depending on their performance and to carry out genotyping of cattle for the SNPs rs137396952 and rs134055603, for which a high degree of association with the development of ketosis was shown in previous GWAS studies. Utilized genotyping methods include TaqMan and High-Resolution Melt Analysis; genotype analysis was performed using the SNPStats web tool. When comparing the results of genotyping using these technologies, the specifity of allelic discrimination carried out using these methods was demonstrated. Testing of the genotyping results had shown that rs134055603 does not obey the Hardy-Weinberg equilibrium in the studied cohort of animals. Scientific novelty. Obtained genotyping results will be used in further association tests with physiologically valuable parameters of dairy cattle, including resistance to diseases.

BCR::ABL1-like acute lymphoblastic leukaemia: a single institution experience on identification of potentially therapeutic targetable cases

BackgroundBCR::ABL1-like acute lymphoblastic leukaemia (BCR::ABL1-like ALL) is characterized by inferior outcomes. Current efforts concentrate on the identification of molecular targets to improve the therapy results. The accessibility to next generation sequencing, a recommended diagnostic method, is limited. We present our experience in the BCR::ABL1-like ALL diagnostics, using a simplified algorithm.ResultsOut of 102 B-ALL adult patients admitted to our Department in the years 2008–2022, 71 patients with available genetic material were included. The diagnostic algorithm comprised flow cytometry, fluorescent in-situ hybridization, karyotype analysis and molecular testing with high resolution melt analysis and Sanger Sequencing. We recognized recurring cytogenetic abnormalities in 32 patients. The remaining 39 patients were screened for BCR::ABL1-like features. Among them, we identified 6 patients with BCR::ABL1-like features (15.4%). Notably, we documented CRLF2-rearranged (CRLF2-r) BCR::ABL1-like ALL occurrence in a patient with long-term remission of previously CRLF2-r negative ALL.ConclusionsAn algorithm implementing widely available techniques enables the identification of BCR::ABL1-like ALL cases in settings with limited resources.

The Effect of DNA Concentration on the HRM Performance in Detecting Jak2 p.V617F Variant in Patients with Myeloproliferative Neoplasms

Introduction: Janus kinase 2 (JAK2) is an intracellular signaling protein. JAK2 p. V617F is a common variant in normal karyotype myeloproliferative neoplasms (MPN). Highresolution melting (HRM) analysis is one of the essential methods for detecting the JAK2 p.V617F variant. In this study, we have investigated the effect of DNA concentration on detecting the JAK2 p.V617F variant using the HRM method. Methods: Genomic DNA was extracted from human blood and diluted ten times in distilled water from 1 to 0.03; afterward, HRM was conducted for each dilution (triplicate). Using SPSS v.20.0 software, the mean Tm of each dilution was calculated and compared. Results: The HRM results revealed the JAK2 wild type and variant to have Tms of 81/64°C and 80/76°C, respectively. At the endpoint of the pre-amplification, the dilutions had different emissions. The statistical analysis revealed no statistically significant differences in Tm between samples with varying DNA concentrations (P value > 0.05). Conclusion: There have been no significant differences obtained in the analysis of JAK2 p.V617F point variant in different DNA dilutions, implying that the HRM analysis has no relation to DNA concentration.

Association between MGMT Enhancer Methylation and MGMT Promoter Methylation, MGMT Protein Expression, and Overall Survival in Glioblastoma.

The repair protein O6-methylguanine-DNA methyltransferase (MGMT) is regulated epigenetically, mainly by the methylation of the MGMT promoter. MGMT promoter methylation status has emerged as a prognostic and predictive biomarker for patients with newly diagnosed glioblastoma (GBM). However, a strong negative correlation between MGMT promoter methylation and MGMT protein expression cannot be applied as a rule for all GBM patients. In order to investigate if the DNA methylation status of MGMT enhancers is associated with MGMT promoter methylation, MGMT expression, and the overall survival (OS) of GBM patients, we established assays based on high-resolution melting analysis and pyrosequencing for one intragenic and three intergenic MGMT enhancers. For CpGs in an enhancer located 560 kb upstream of the MGMT promoter, we found a significant negative correlation between the methylation status and MGMT protein levels of GBM samples expressing MGMT. The methylation status of CpGs in the intragenic enhancer (hs696) was strongly negatively correlated with MGMT promoter methylation and was significantly higher in MGMT-expressing GBM samples than in MGMT-non-expressing GBM samples. Moreover, low methylation of CpGs 01-03 and CpGs 09-13 was associated with the longer OS of the GBM patients. Our findings indicate an association between MGMT enhancer methylation and MGMT promoter methylation, MGMT protein expression, and/or OS.

Toxoplasma gondii genotyping in AIDS patients using high resolution melt analysis (HRM analysis) in Khuzestan province, southwest Iran.

Toxoplasmosis is a parasitic zoonotic disease caused by Toxoplasma gondii, a common protozoan in the Apicomplexa phylum. Several studies in Iran have demonstrated the presence of the parasite in various hosts, but no data on T. gondii genotyping in HIV patients in Khuzestan, Southwest Iran, is available. One hundred of blood samples from AIDS patients were collected and tested by real-time PCR High Resolution Melting analyses for T.gondii detection and genotyping. T. gondii was discovered in 8 out of 100 (8%) AIDS patients with dominant Type I. This study suggest that HRM method demonstrated excellent discriminating ability for T. gondii, and AIDS patients should be tested for Toxoplasma detection and genotyping to prevent parasite pathogenicity.

Three major chlorotype lineages in chamomile (Matricaria chamomilla L., Asteraceae)

Chamomile (Matricaria chamomilla L., Asteraceae) is one of the most important medicinal plants with antiphlogistic, spasmolytic, carminative, antibacterial and antimycotic properties. Thirty-one chloroplast markers were developed, optimised for high-resolution melting analysis. Subsequently, 23 M. chamomilla accessions (247 individuals) complemented with 2 individuals of the closely related species M. discoidea were analysed with this marker set. The marker set can practically be reduced to 20 markers without information loss due to the linkage of 16 markers. In total, 20 chlorotypes (multilocus genotypes) were identified, organised in three evolutionary main lineages. Only 8 accessions were monomorphic, the other 15 accessions had between 2 and 6 chlorotypes per accession. The high number of polymorphic accessions and the high number of chlorotypes within many accessions indicate already a high degree of variability within accessions, confirmed by the 66% variation within by AMOVA. Gene diversity of the polymorphic accessions ranged between 0.069 and 0.261. Since many cultivars had their origin in wild populations of the countries where breeding took place, a first hypothetical indication of the geographic origin of chamomile could point to Albania and adjacent countries. The ‘ancient chlorotype’ was identified by comparing published plastome sequences of Artemisia annua and Lactuca sativa. The ‘ancient chlorotype’ was present only in the closely related M. discoidea but not in any of the M. chamomilla accessions. Two commercially important tetraploid cultivars proofed to be unrelated to their presumed diploid parents.

High-Resolution Melting (HRM) Analysis for Rapid Molecular Identification of Sparidae Species in the Greek Fish Market.

The red porgy (Pagrus pagrus) and the common dentex (Dentex dentex) are Sparidae species of high commercial value, traded in the Greek market. In some cases, fish species identification from Greek fisheries is difficult for the consumer due to the strong morphological similarities with their imported counterparts or closely related species such as Pagrus major, Pagrus caeroleustictus, Dentex gibbosus and Pagellus erythrinus, especially when specimens are frozen, filleted or cooked. Techniques based on DNA sequencing, such as COI barcoding, accurately identify species substitution incidents; however, they are time consuming and expensive. In this study, regions of mtDNA were analyzed with RFLPs, multiplex PCR and HRM in order to develop a rapid method for species identification within the Sparidae family. HRM analysis of a 113 bp region of cytb and/or a 156 bp region of 16s could discriminate raw or cooked samples of P. pagrus and D. dentex from the aforementioned closely related species and P. pagrus specimens sampled in the Mediterranean Sea when compared to those fished in the eastern Atlantic. HRM analysis exhibited high accuracy and repeatability, revealing incidents of mislabeling. Multiple samples can be analyzed within three hours, rendering this method a useful tool in fish fraud monitoring.

Molecular detection and identification of Diatrypaceous airborne spores in Australian vineyards revealed high species diversity between regions.

The grapevine trunk disease, Eutypa dieback (ED), causes significant vine decline and yield reduction. For many years, the fungus Eutypa lata was considered the main pathogen causing ED of grapevines in Australia. Recent studies showed other Diatrypaceous fungi were also associated with vines exhibiting dieback symptoms but there is limited information on how these fungal pathogens spread in vineyards. Thus, information on the spore dispersal patterns of Diatrypaceous fungi in different wine regions will assist in identifying high-risk infection periods in vineyards. Using more than 6800 DNA samples from airborne spores collected from eight wine regions in south-eastern Australia over 8 years using a Burkard spore trap, this study investigated the diversity and abundance of Diatrypaceous species, using multi-faceted molecular tools. A multi-target quantitative PCR (qPCR) assay successfully detected and quantified Diatrypaceous spores from 30% of the total samples with spore numbers and frequency of detection varying between regions and years. The high-resolution melting analysis (HRMA) coupled with DNA sequencing identified seven species, with E. lata being present in seven regions and the most prevalent species in the Adelaide Hills, Barossa Valley and McLaren Vale. Cryptovalsa ampelina and Diatrype stigma were the predominant species in the Clare Valley and Coonawarra, respectively while Eutypella citricola and Eu. microtheca dominated in the Hunter Valley and the Riverina regions. This study represents the first report of D. stigma and Cryptosphaeria multicontinentalis in Australian vineyards. This study further showed rainfall as a primary factor that triggers spore release, however, other weather factors that may influence the spore release in different climatic regions of Australia still requires further investigation.

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Focus Issue Articles on Diagnostic Assay Development and Validation: The Science of Getting It Right

Associations between Genetic Variants in DAB, PRKAG, and DACH Genes and Gender in Chronic Kidney Disease

Background: Recent genome-wide association studies demonstrated the association between the prevalence of chronic kidney disease (CKD) and rs11959928, rs626277, and rs7805747 polymorphisms. Materials and Methods: In this study, we investigated the association between CKD and these polymorphisms in patients and controls according to gender. High-resolution melting analysis was performed to detect DAB2 rs11959928, DACH1 rs626277, and PRKAG2 rs7805747 single nucleotide polymorphisms. Genomic DNA was extracted from the buffy coat of 163 patients with chronic renal disease and 218 control individuals. Ten percent of the results were also randomly confirmed by direct DNA sequencing. Results: Multivariable logistic regression analysis with adjustment for confounders showed rs7805747 (dominant model) has a statistically significant protective effect in females, and rs11959928 (additive and dominant models) was significantly associated with the prevalence of CKD in males. rs7805747 (recessive model) was significantly associated with the prevalence of CKD in males. Conclusion: The very same genetic variants have different effects in males and females separately. Our results warrant the need for similar studies in larger cohorts.

Rapid Detection of Predominant SARS-CoV-2 Variants Using Multiplex High-Resolution Melting Analysis.

Coronavirus disease 2019, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a considerable threat to global public health. This study developed and evaluated a rapid, low-cost, expandable, and sequencing-free high-resolution melting (HRM) assay for the direct detection of SARS-CoV-2 variants. A panel of 64 common bacterial and viral pathogens that can cause respiratory tract infections was employed to evaluate our method's specificity. Serial dilutions of viral isolates determined the sensitivity of the method. Finally, the assay's clinical performance was assessed using 324 clinical samples with potential SARS-CoV-2 infection. Multiplex HRM analysis accurately identified SARS-CoV-2 (as confirmed with parallel reverse transcription-quantitative PCR [qRT-PCR] tests), differentiating between mutations at each marker site within approximately 2 h. For each target, the limit of detection (LOD) was lower than 10 copies/reaction (the LOD of N, G142D, R158G, Y505H, V213G, G446S, S413R, F486V, and S704L was 7.38, 9.72, 9.96, 9.96, 9.50, 7.80, 9.33, 8.25, and 8.25 copies/reaction, respectively). No cross-reactivity occurred with organisms of the specificity testing panel. In terms of variant detection, our results had a 97.9% (47/48) rate of agreement with standard Sanger sequencing. The multiplex HRM assay therefore offers a rapid and simple procedure for detecting SARS-CoV-2 variants. IMPORTANCE In the face of the current severe situation of increasing SARS-CoV-2 variants, we developed an upgraded multiplex HRM method for the predominant SARS-CoV-2 variants based on our original research. This method not only could identify the variants but also could be utilized in subsequent detection of novel variants since the assay has great performance in terms of flexibility. In summary, the upgraded multiplex HRM assay is a rapid, reliable, and economical detection method, which could better screen prevalent virus strains, monitor the epidemic situation, and help to develop measures for the prevention and control of SARS-CoV-2.

Differential detection of sheep and goat meat using duplex real-time PCR and high-resolution melt analysis

Authenticity in meat and meat products is of paramount importance in preventing adulteration and ensuring food safety. Since morphologically indistinct samples like sheep and goat meat requires accurate and sensitive differentiation, we developed and validated a duplex real-time polymerase chain reaction (qPCR) assay with high-resolution melt analysis (HRMA) for simultaneous identification of sheep and goat meat. Species-specific amplicons with melting temperature of 78 °C for goat and 80 °C for sheep (with associated standard errors) was identified. The assay was optimized for basic PCR parameters and the HRMA detection was standardized based on melt curve shape, color and inflexion points. The absolute limit of detection (LODabs) and limit of quantification (LOQ) of the assay were found to be 0.39 ng and 0.78 ng of target DNA respectively. A sensitivity of 0.1% was obtained for both sheep and goat meat. The relative limit of detection (LODrel) was also found to be 5%. The linearity and efficiency were evaluated in both simplex and duplex assays. The assay applicability was evaluated using proficiency test samples and commercially available raw and processed meat samples. The obtained results showed that this closed-tube qPCR-HRMA method can be a rapid and sensitive detection technique for meat speciation.

Trypanosomes infection, endosymbionts, and host preferences in tsetse flies (Glossina spp.) collected from Akagera park region, Rwanda: A correlational xenomonitoring study

Akagera National Park and its surroundings are home to tsetse flies and a number of their mammalian hosts in Rwanda. A One-health approach is being used in the control and surveillance of both animal and human trypanosomosis in Rwanda. Determination of the infection level in tsetse flies, species of trypanosomes circulating in vectors, the source of tsetse blood meal and endosymbionts is crucial in understanding the epidemiology of the disease in animals and humans in the region.Tsetse flies (n = 1101), comprising Glossina pallidipes (n = 771) and Glossina morsitans centralis (n = 330) were collected from Akagera park and surrounding areas between May 2018 and June 2019. The flies were screened for trypanosomes, vertebrate host DNA to identify sources of blood meal, and endosymbionts by PCR - High Resolution Melting analysis and amplicon sequencing. The feeding frequency and the feeding indices (selection index - W) were calculated to identify the preferred hosts. An overall trypanosome infection rate of 13.9% in the fly's Head and Proboscis (HP) and 24.3% in the Thorax and Abdomen (TA) were found. Eight trypanosome species were identified in the tsetse fly HP and TA, namely: Trypanosoma (T.) brucei brucei, T. congolense Kilifi, T. congolense savannah, T. vivax, T. simiae, T. evansi, T. godfreyi, T. grayi and T. theileri. We found no evidence of human-infective T. brucei rhodesiense. We also identified eighteen species of vertebrate hosts that tsetse flies fed on, and the most frequent one was the buffalo (Syncerus caffer) (36.5%). The frequently detected host by selection index was the rhinoceros (Diceros bicornis) (W = 16.2). Most trypanosome infections in tsetse flies were associated with the buffalo blood meal. The prevalence of tsetse endosymbionts Sodalis and Wolbachia was 2.8% and 4.8%, respectively. No Spiroplasma and Salivary Gland Hypertrophy Virus were detected. These findings implicate the buffaloes as the important reservoirs of tsetse-transmitted trypanosomes in the area. This contributes to predicting the main cryptic reservoirs and therefore guiding the effective control of the disease. The study findings provide the key scientific information that supports the current One Health collaboration in the control and surveillance of tsetse-transmitted trypanosomosis in Rwanda.

The impact of PIK3CA mutations and PTEN expression on the effect of neoadjuvant therapy for postmenopausal luminal breast cancer patients

BackgroundThere is pressing needs to find the biomarker in the selection of neoadjuvant therapy in postmenopausal luminal breast cancer patients. We examined the hypothesis that PIK3CA mutations and low phosphatase and tensin homolog (PTEN) expression affect the response to neoadjuvant therapy and prognosis in postmenopausal luminal breast cancer patients.MethodsPostmenopausal patients with estrogen receptor-positive, human epidermal growth factor receptor 2-negative breast cancer, up to stage II, who underwent neoadjuvant chemotherapy (NAC; n = 60) or neoadjuvant endocrine therapy (NAE; n = 55) were selected. PIK3CA exon 9 and exon 20 mutations were screened by high resolution melting analysis and confirmed by Sanger sequence. PTEN expression was evaluated by immunohistochemistry. The relationships among PIK3CA mutations, PTEN expression, clinicopathological features, the pathological effect of neoadjuvant therapy, recurrence-free survival (RFS) and overall survival were analyzed.ResultsAmong 115 patients, PIK3CA mutations and low PTEN expression before treatment were detected in 35 patients (30.4%) and in 28 patients (24.3%), respectively. In the NAC group, tumor with PIK3CA mutations showed significantly poorer response than tumor with PIK3CA wild-type (p = 0.03). On the other hand, in the NAE group, there was no significant difference in pathological therapeutic effect between tumor with PIK3CA mutations and tumor with PIK3CA wild-type (p = 0.54). In the NAC group, the log-rank test showed no difference in RFS between patients with PIK3CA mutations and PIK3CA wild-type (p = 0.43), but patients with low PTEN expression showed significantly worse RFS compared to patients with high PTEN expression (5 year RFS 0.64 vs. 0.87, p = 0.01). In the Cox proportional hazards model for RFS, PTEN expression, progesterone receptor, and pathological therapeutic effect were predictive factors for time to recurrence (All p < 0.05).ConclusionsPIK3CA mutations are associated with resistance to NAC but do not affect the response to NAE. Low PTEN expression does not affect response to either NAC or NAE but correlates with shorter RFS in patients who received NAC. These biomarkers will be further evaluated for clinical use to treat postmenopausal luminal breast cancer patients.

Potential use of high-resolution melting analyses for SARS-CoV-2 genomic surveillance

The pandemic caused by COVID-19 and the emergence of new variants of SARS-CoV-2 have generated clinical and epidemiological impacts on a global scale. The use of strategies for monitoring viral circulation and identifying mutations in genomic regions involved in host interaction are important measures to mitigate viral dissemination and reduce its likely complications on population health. In this context, the objective of this work was to explore the potential of high-resolution melting (HRM) analysis combined with one-step real-time reverse transcription PCR in a closed-tube system, as a fast and convenient method of screening for SARS-CoV-2 mutations with possible implications on host-pathogen interactions. The HRM analyses allowed the distinction of the Gamma, Zeta, Alpha, Delta, and Omicron variants against the predecessors (B.1.1.28, B.1.1.33) of occurrence in Brazil. It is concluded that the molecular tool standardized here has the potential to optimize the genomic surveillance of SARS-CoV-2, and could be adapted for genomic surveillance of other pathogens, due to its ability to detect, prior to sequencing, samples suggestive of new variants, selecting them more assertively and earlier for whole genome sequencing when compared to random screening.

Combining the amplification refractory mutation system and high-resolution melting analysis for KRAS mutation detection in clinical samples

The success of personalized medicine depends on the discovery of biomarkers that allow oncologists to identify patients that will benefit from a particular targeted drug. Molecular tests are mostly performed using tumor samples, which may not be representative of the tumor’s temporal and spatial heterogeneity. Liquid biopsies, and particularly the analysis of circulating tumor DNA, are emerging as an interesting means for diagnosis, prognosis, and predictive biomarker discovery. In this study, the amplification refractory mutation system (ARMS) coupled with high-resolution melting analysis (HRMA) was developed for detecting two of the most relevant KRAS mutations in codon 12. After optimization with commercial cancer cell lines, KRAS mutation screening was validated in tumor and plasma samples collected from patients with pancreatic ductal adenocarcinoma (PDAC), and the results were compared to those obtained by Sanger sequencing (SS) and droplet digital polymerase chain reaction (ddPCR). The developed ARMS-HRMA methodology stands out for its simplicity and reduced time to result when compared to both SS and ddPCR but showing high sensitivity and specificity for the detection of mutations in tumor and plasma samples. In fact, ARMS-HRMA scored 3 more mutations compared to SS (tumor samples T6, T7, and T12) and one more compared to ddPCR (tumor sample T7) in DNA extracted from tumors. For ctDNA from plasma samples, insufficient genetic material prevented the screening of all samples. Still, ARMS-HRMA allowed for scoring more mutations in comparison to SS and 1 more mutation in comparison to ddPCR (plasma sample P7). We propose that ARMS-HRMA might be used as a sensitive, specific, and simple method for the screening of low-level mutations in liquid biopsies, suitable for improving diagnosis and prognosis schemes.Graphical

Rapid Detection and Identification of Fasciola spp. and Dicrocoelium spp. Isolated from the Ruminant Livestock of Northwest Iran Using High-Resolution Melting Analysis (HRM).

The liver flukes of the Fasciola species and Dicrocoelium spp. are recognised as parasites of domestic and wild herbivores. Both species of F. hepatica and F. gigantica as well as D. dendriticum are distributed in Iran. The present study aimed to identify Fasciola spp. and Dicrocoelium spp. using mitochondrial Cox1 (cytochrome c oxidase I) gene by HRM method. Totally, thirty infected liver specimens were collected from the sheep (n:23) and cattle (n:7) at the abattoirs of Qazvin Province, northwest Iran in 2022. DNA extraction and PCR amplification of Cox1 gene were conducted by HRM technique. DnaSP v.5.0 was used for compression of diversity indices of ribosomal 28S rDNA and mitochondrial Cox1 markers of Dicrocoelium spp. The taxonomic status of Dicrocoelium spp. was performed by sequencing and phylogenetic analysis. Overall, 26 and 4 isolates were identified as F. hepatica and F. gigantica, respectively. D. dendriticum was the sole infecting species of Dicrocoelium revealed by HRM analysis. Genomic analysis showed a moderate (28S rDNA genes: 0.600±0.215) to high (Cox1: 0.733±0.155) haplotype diversity for D. dendriticum. The parasite-dependent mitochondrial gene (Cox1) could identify a higher genetic diversity of D. dendriticum compared to nuclear 28S rDNA gene. HRM technique in the present study found to be a reliable technique for identification and genetic diversity of liver flukes but more comprehensive and in-depth studies in different parts of the country are needed.

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

The genus Thunnus (family Scombridae) comprises eight species of tunas of which all but one are targeted by industrialized fisheries. While 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 molecular species identification. Here we investigate short amplicon (SA) and unlabeled probe (UP) high-resolution melting analysis (HRMA) as a low-cost, high-throughput molecular genotyping assay capable of distinguishing between albacore tuna (T. alalunga), blackfin tuna (T. atlanticus), bigeye tuna (T. obesus), Atlantic bluefin tuna (T. thynnus), and yellowfin tuna (T. albacares) in the Gulf of Mexico. While SA-HRMA of variable regions in the NADH dehydrogenase subunit 4 (ND4) and subunit 5 (ND5), and subunit 6 (ND6) of the mitochondrial DNA (mtDNA) genome did yield some species-specific diagnostic melting curves (ex. 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°C, 62°C, 59°C, 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.

High resolution melting analysis: A rapid and sensitive method for detection of mutations induced by CRISPR/Cas9-based genome editing in oysters

Genome editing using CRISPR/Cas9 methodology holds much potential to accelerate the genetic improvement in aquaculture, and has been extensively tested and applied to various aquaculture species. However, screening and identifying successfully edited individuals based on sensitive, low-cost, and rapid methods remains challenging. Here, we developed and optimized a high-resolution melting analysis (HRM) assay to screen CRISPR/Cas9-edited larval oysters. The target amplification size range was optimized to 100–200 bp. Compared with fluorescent dyes SYBR Green and SYTO-9, saturated dye EvaGreen showed high stability and repeatability when used in PCR master mixes. The HRM assay was sensitive enough to detect a temperature shift when a mixture of 10% mutant colonies and 90% WT colonies were present. Sanger sequencing analysis confirmed that oysters with indel mutations could be successfully identified by the HRM method. Together, these data indicate that the HRM assay developed in this study is a reproducible, sensitive, and efficient method for rapidly screening and identifying gene-edited oysters.

The Application of High-Resolution Melting Analysis to trnL (UAA) Intron Allowed a Qualitative Identification of Apple Juice Adulterations

Food authenticity plays a pivotal role in the modern age since an increased consumers awareness has led them to pay more attention to food commodities. For this reason, it is important to have reliable and fast techniques able to detect possible adulterations in food, which affect qualitative and economic value. Therefore, the aim of this study was to detect possible adulterations in apple juice from others fruit species (i.e., pear, peach, and kiwi) combining DNA barcoding approach, using trnL (UAA) intron, with high resolution melting analysis (HRMA). A preliminary phylogenetic analysis, using sequences retrieved by the GenBank, confirmed the discriminatory power of trnL (UAA) intron among the four fruit species examined. Moreover, the sequencing of the trnL (UAA) fragments obtained from apple, pear, peach, and kiwi, demonstrated the suitability of an inner shorter sequence, P6 loop, to differentiate the considered species. The HRMA coupled with trnL (UAA) intron allowed discrimination among the four fruits but provided incomplete results for juices. Whereas the HRMA targeting the P6 loop amplicons confirmed the suitability of the technique to qualitatively distinguish fruit juices composed by the combination of apple/pear and apple/peach. However, the impossibility of discriminating apple/kiwi juices from the pure kiwi sample highlighted limitations, most likely related to the DNA extraction process. This hypothesis was further confirmed by analyzing DNA blends obtained by combining nucleic acids extracted from pure matrixes (i.e., apple and kiwi fruits). In this specific case, the application of HRMA allowed both qualitative and quantitative assessment of the samples.