High-resolution Melting Assay Research Articles

Distinguishing venomous jellyfish species via high resolution melting analysis

Accurate species identification is critical for highly venomous jellyfish studies particularly for evaluation of their distribution as it is important not only for understanding population dynamics but also predicting risk to locals and tourists. Hazardous jellyfish species have been determined using methods such as comparative morphology and chemistry, which are less useful and practical in some cases. A few molecular methods have been developed in aiding hazardous jellyfish species identification. However, no studies have focused on designing and developing high resolution melting (HRM) assays to distinguish jellyfish species from each other. This is despite several studies showing the potential of HRM in effectively distinguishing plants and animals. In this study, we developed an HRM method to differentiate jellyfish species in five box jellyfish families (Carukiidae, Chirodropidae, Chiropsalmidae, Chiropsellidae, and Tripedaliidae) and Physaliidae found in Thai waters. All jellyfish samples (10 species) were able to be distinguished by the HRM with 16S rRNA primers. Even closely related species in the same genus could be clearly discriminated by their melting profiles. The melting curves of the 10 species were then used as references. By comparing the normalized melting curves of twenty-two jellyfish specimens with the reference curves, the tested specimens were assigned into species. In addition, HRM results of all jellyfish specimens tested in this study were similar to those of morphological based identification by experts. This is the first attempt of using HRM analysis to determine species of jellyfish collected from Thai waters.

Multiplex high-resolution melting assay for simultaneous detection of five key bacterial pathogens in urinary tract infections: A pilot study.

The diagnosis of urinary tract infections (UTIs) is usually based on the results of urine culture, but it is time-consuming, labor-intensive and has a low sensitivity. The aim of this study was to develop multiplex high-resolution melting assay (MHRM) for the simultaneous detection of five common bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Staphylococcus saprophyticus, Enterococcus faecalis, and group B streptococci (GBS)) directly from urine samples. A total of 287 urine specimens were evaluated by HRM assay and the results were compared with the conventional culture method. Five different melt curves generated and differentiated five bacterial pathogens. The detection limit of the MHRM assay was 1.5 × 103 CFU/ml for E. coli and K. pneumoniae and 1.5 × 102 CFU/ml for S. saprophyticus, E. faecalis and GBS. Compared to culture, the specificity of the MHRM assay ranged from 99.3 to 100%, and sensitivity 100% for all test pathogens. The MHRM assay developed in the current study might be functional tool for the diagnosis of UTIs and has the potential for direct detection of the organism in the clinical samples. Additionally, it creates results in less than 5 h, helping clinicians to start treatment with appropriate antimicrobial agents. This method could be a useful supplement to urine culture.

Cytochrome P450 2D6 (CYP2D6) and glucose-6-phosphate dehydrogenase (G6PD) genetic variations in Thai vivax malaria patients: Implications for 8-aminoquinoline radical cure.

Primaquine and tafenoquine are the only licensed drugs that effectively kill the hypnozoite stage and are used to prevent Plasmodium vivax malaria relapse. However, both primaquine and tafenoquine can cause acute hemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient people with varying degrees of severity depending on G6PD variants. Additionally, primaquine efficacy against malaria parasites was decreased in individuals with impaired cytochrome P450 2D6 (CYP2D6) activity due to genetic polymorphisms. This study aimed to characterize G6PD and CYP2D6 genetic variations in vivax malaria patients from Yala province, a malaria-endemic area along the Thai-Malaysian border, and determine the biochemical properties of identified G6PD variants. Multiplexed high-resolution melting assay and DNA sequencing detected five G6PD variants, including G6PD Kaiping, G6PD Vanua Lava, G6PD Coimbra, G6PD Mahidol, and G6PD Kerala-Kalyan. Biochemical and structural characterization revealed that G6PD Coimbra markedly reduced catalytic activity and structural stability, indicating a high susceptibility to drug-induced hemolysis. While Kerala-Kalyan had minor effects, it is possible to develop mild adverse effects when receiving radical treatment. CYP2D6 genotyping was performed using long-range PCR and DNA sequencing, and the phenotypes were predicted using the combination of allelic variants. Decreased and no-function alleles were detected at frequencies of 53.4% and 14.2%, respectively. The most common alleles were CYP2D6*36+*10 (25.6%), *10 (23.9%), and *1 (22.2%). Additionally, 51.1% of the intermediate metabolizers showed CYP2D6*10/*36+*10 as the predominant genotype (15.9%). Our findings provide insights about genetic variations of G6PD and CYP2D6 in 88 vivax malaria patients from Yala, which may influence the safety and effectiveness of radical treatment. Optimization of 8-aminoquinoline administration may be required for safe and effective treatment in the studied population, which could be a significant challenge in achieving the goal of eliminating malaria.

TBX 5 gene mutation analysis among Tanzanian children with congenital heart diseases using high-resolution melting assays

Early cardiac development is governed by transcription factor genes. TBX5, a T-box transcription factor gene, plays an important role in the development of the second heart field during cardiac septation by promoting cell cycle progression through the enhancement of Cdk6 and hedgehog signaling pathways. TBX5 binds to the promoter region of genes, enhancing the expression of alpha cardiac myosin heavy chain 6 (MYH6), which is a predominant isoform found in human cardiac tissue. TBX5 gene mutations are postulated to cause congenital heart diseases. A casecontrol TBX5 mutational analysis was performed to provide insight into the etiology of sporadic congenital heart diseases in our setting. We used a magnetic induction cycler (mic-PCR), which is a next-generation tool for polymerase chain reaction-high resolution melting assays, to detect mutations in children with sporadic isolated congenital heart diseases. A retrospective casecontrol study was conducted at the Jakaya Kikwete Cardiac Institute. The peripheral blood samples were collected, and DNA was extracted using the Quick-DNA Miniprep Kit. The primers were designed using Primer 3 software, validated using the program BLAST, and checked for hairpin and homo-hetero-dimerization using the IDT oligo analyzer. Real-time polymerase chain reaction (PCR)-high-resolution melting assays for screening TBX5 gene mutations were done using a magnetic induction cycler. We found two (2) TBX5 mutations in exon 5, among patients with Atrial-Ventral Septal Defects (ASVD) and Atrial-Septal Defects (ASD) and none among controls. TBX5 exon 5 is a molecular hotspot for isolated congenital heart diseases.

The Development of a Real-Time PCR Assay for Specific Detection of the NISKHI Sheep Pox Vaccine Virus Strain DNA

Sheep pox (SPP) constitutes a global animal health scourge, despite the numerous efforts targeting the eradication of the disease implemented in affected countries. An efficient control and eradication strategy incorporates the use of live attenuated vaccines, which in turn requires a method for differentiation between vaccinated and infected sheep. The NISKHI live attenuated SPP vaccine (LAV) is abundantly used in Russia, Kazakhstan and other Central Asian countries. This study describes the development and evaluation of a real-time PCR with a high-resolution melting assay, capable of differentiating the NISKHI vaccine virus from circulating virulent field strains. The RNA polymerase subunit RPO132 gene contains a unique single nucleotide polymorphism (SNP) capable of altering the melting curves of amplicons from LAV and virulent field isolates circulating in the region. The melting temperature (Tm) of field isolates ranged from 75.47 °C ± 0.04 to 75.86 °C ± 0.08, while the vaccine strain averaged 76.46 °C ± 0.12. Subsequent evaluation of this assay demonstrated that the recent SPP outbreaks in central Russia may be attributed to virulent field isolates. This robust assay was proven to consistently and differentially detect the NISKHI LAV strain when analyzing clinical samples from affected sheep.

Design and Implementation of a Dual-Region Self-Referencing Fiber-Optic Surface Plasmon Resonance Biosensor.

The need for self-referencing is extremely important in the field of biosensing. In this manuscript, we report on the study, design, and validation of a dual-region self-referencing fiber-optic surface plasmon resonance biosensor. One region is intended to measure and monitor the binding events of the biological sample under test, while the other one is designed to be used as a reference channel to compensate for external factors, such as bulk refractive index modifications and temperature oscillations, that can negatively affect the biomolecular interaction measurement. Two different configurations for the biosensor probe are presented and investigated here, both theoretically and experimentally. First, the theoretical performance of the proposed biosensor probes, in terms of surface plasmon resonance wavelength shift, was simulated using a numerical model. Afterward, they were experimentally validated in sucrose-water solutions and showed a response to refractive index and temperature changes with sensitivities up to 2000 nm/RIU and 1.559 nm/°C, respectively. Finally, an aptamer-based bioassay and a high-resolution melting assay were successfully implemented on the two proposed configurations, demonstrating the feasibility of analyzing the binding events and measuring other external signal modifications simultaneously using the same biosensor probe.

Seasonal Dynamics of Flight Phenology of the Euwallacea fornicatus Species Complex and an Associated Parasitoid Wasp in Avocado Groves in Taiwan.

The Euwallacea fornicatus species complex (Coleoptera: Curculionidae: Scolytinae: Xyleborini) is a group of four cryptic ambrosia beetle species. Native to Asia, several members of the complex have invaded other continents, where they cause significant economic losses to agricultural crops (e.g., avocado) and natural ecosystems. We were primarily interested in developing management strategies by focusing on the flight behavior of the beetles. Thus, seasonal differences in flight activity were assessed using panel traps baited with a commercial quercivorol lure, placed in infested avocado orchards in Danei, Tainan, Taiwan. Same traps were used to investigate the flight activity of a natural enemy, an undescribed species of the Braconid genus Eucosmophorus sp. Shothole borer species were identified using a DNA-based, high resolution melting assay. Trap data were compared to the predictions of a simple degree-day model, incorporating developmental data and several environmental parameters known to influence flight. Such as the time period representing most of flight activity in a day and temperature-dependent flight propensity. In stark contrast to the degree-day model which predicted the highest emergence, and by extension flight, of shothole borers during spring and summer (May to November), flight activity was actually lowest during these months, and instead, peaked during the winter (October to March). Abundance of the parasitoid wasp closely mirrored flight activity of the shothole borers. The mismatch of trapping and modeling data can have many causes, heavy precipitation and possibly cooperative brood care may suppress the dispersal behavior of the shothole borers during the summer.

Hypervariable-Locus Melting Typing: a Novel Approach for More Effective High-Resolution Melting-Based Typing, Suitable for Large Microbiological Surveillance Programs.

ABSTRACTPathogen typing is pivotal to detecting the emergence of high-risk clones in hospital settings and to limit their spread. Unfortunately, the most commonly used typing methods (i.e., pulsed-field gel electrophoresis [PFGE], multilocus sequence typing [MLST], and whole-genome sequencing [WGS]) are expensive or time-consuming, limiting their application to real-time surveillance. High-resolution melting (HRM) can be applied to perform cost-effective and fast pathogen typing, but developing highly discriminatory protocols is challenging. Here, we present hypervariable-locus melting typing (HLMT), a novel approach to HRM-based typing that enables the development of more effective and portable typing protocols. HLMT types the strains by assigning them to melting types (MTs) on the basis of a reference data set (HLMT-assignment) and/or by clustering them using melting temperatures (HLMT-clustering). We applied the HLMT protocol developed on the capsular gene wzi for Klebsiella pneumoniae on 134 strains collected during surveillance programs in four hospitals. Then, we compared the HLMT results to those obtained using wzi, MLST, WGS, and PFGE typing. HLMT distinguished most of the K. pneumoniae high-risk clones with a sensitivity comparable to that of PFGE and MLST+wzi. It also drew surveillance epidemiological curves comparable to those obtained using MLST+wzi, PFGE, and WGS typing. Furthermore, the results obtained using HLMT-assignment were consistent with those of wzi typing for 95% of the typed strains, with a Jaccard index value of 0.9. HLMT is a fast and scalable approach for pathogen typing, suitable for real-time hospital microbiological surveillance. HLMT is also inexpensive, and thus, it is applicable for infection control programs in low- and middle-income countries.IMPORTANCE In this work, we describe hypervariable-locus melting typing (HLMT), a novel fast approach to pathogen typing using the high-resolution melting (HRM) assay. The method includes a novel approach for gene target selection, primer design, and HRM data analysis. We successfully applied this method to distinguish the high-risk clones of Klebsiella pneumoniae, one of the most important nosocomial pathogens worldwide. We also compared HLMT to typing using WGS, the capsular gene wzi, MLST, and PFGE. Our results show that HLMT is a typing method suitable for real-time epidemiological investigation. The application of HLMT to hospital microbiology surveillance can help to rapidly detect outbreak emergence, improving the effectiveness of infection control strategies.

Discrimination of SARS-CoV-2 Omicron Sublineages BA.1 and BA.2 Using a High-Resolution Melting-Based Assay: a Pilot Study.

ABSTRACTThe Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. As of March 2022, Omicron variant BA.2 is rapidly replacing variant BA.1. As variant BA.2 may cause more severe disease than variant BA.1, variant BA.2 requires continuous monitoring. The current study aimed to develop a novel high-resolution melting (HRM) assay for variants BA.1 and BA.2 and to determine the sensitivity and specificity of our method using clinical samples. Here, we focused on the mutational spectra at three regions in the spike receptor-binding domain (RBD; R408, G446/L452, and S477/T478) for the variant-selective HRM analysis. Each variant was identified based on the mutational spectra as follows: no mutations (Alpha variant); L452R and T478K (Delta variant); G446S and S477N/T478K (Omicron variant BA.1); and R408S and S477N/T478K (Omicron variant BA.2). Upon analysis of mutation-coding RNA fragments, the melting curves of the wild-type fragments were distinct from those of the mutant fragments. The sensitivity and specificity of this method were determined as 100% and more than 97.5%, respectively, based on 128 clinical samples (40 Alpha, 40 Delta, 40 Omicron variant BA.1/BA.1.1, and 8 Omicron variant BA.2). These results suggest that this HRM-based assay is a promising screening method for monitoring the transmission of Omicron variants BA.1 and BA.2.IMPORTANCE This study seeks to apply a novel high-resolution melting (HRM) assay to identify and discriminate BA.1 and BA.2 sublineages of the SARS-CoV-2 Omicron variant. Variant BA.2 may cause more severe disease than variant BA.1, meaning that identifying this variant is an important step toward improving the care of patients suffering from COVID-19. However, screening for these variants remains difficult, as current methods mostly rely on next-generation sequencing, which is significantly costlier and more time-consuming than other methods. We believe that our study makes a significant contribution to the literature because we show that this method was 100% sensitive and over 97.5% specific in our confirmation of 128 clinical samples.

Simultaneous Detection of Foodborne Pathogens Using a Real-Time PCR Triplex High-Resolution Melt Assay

Foodborne pathogens pose risks to populations all over the world. Pathogens can be used as bioterrorism agents, causing an outbreak that affects many individuals through the consumption of a commonly affected food or beverage. A PCR assay can be used to identify pathogens through their unique melting points using a high-resolution melt assay. Assays can be used to detect the bacteria individually or from a mixture using species-specific primers. An assay was developed to detect and identify three pathogens that routinely cause multistate foodborne outbreaks, as documented by the U.S. Centers for Disease Control and Prevention, Campylobacter jejuni (C. jejuni), Escherichia coli (E. coli), and Salmonella enterica (S. enterica), in single bacterium assays and a multiplex. The primers were targeted to specific and unique gene sequences of each pathogen, including cadF, yedN, and hilA, respectively. Each pathogen was identified by its unique melting temperature in single assays: 78.10 ± 0.58 °C for C. jejuni, 81.96 ± 0.42 °C for E. coli, and 87.55 ± 0.37 °C for S. enterica. The multiplex successfully detected and identified all three of the pathogens with the distinctly separated melt peaks. The PCR high-resolution melt assay also proved to be specific, reproducible, fast, and sensitive in experiments.

Identification of Glaesserella parasuis and Differentiation of Its 15 Serovars Using High-Resolution Melting Assays.

Glaesserella parasuis is the etiological agent of Glässer’s disease, which is associated with polyserositis and arthritis and has a significant impact on the economy of the pig production industry. For the optimal surveillance of this pathogen, as well as for the investigation of G. parasuis-associated diseases, it is crucial to identify G. parasuis at the serovar level. In this work, we designed and developed new high-resolution melting (HRM) approaches, namely, the species-specific GPS-HRM1 and two serovar-specific HRM assays (GPS-HRM2 and GPS-HRM3), and evaluated the sensitivity and specificity of the assays. The HRM assays demonstrated good sensitivity, with 12.5 fg–1.25 pg of input DNA for GPS-HRM1 and 125 fg–12.5 pg for GPS-HRM2 and GPS-HRM3, as well as a specificity of 100% for the identification of all recognized 15 G. parasuis serovars. Eighteen clinical isolates obtained between 2014 and 2022 in Switzerland were tested by applying the developed HRM assays, which revealed a heterogeneous distribution of serovars 2, 7, 4, 13, 1, and 14. The combination with virulence marker vtaA (virulence-associated trimeric autotransporters) allows for the prediction of potentially virulent strains. The assays are simple to execute and enable a reliable low-cost approach, thereby refining currently available diagnostic tools.

High-resolution melting (HRM) curve analysis as a potential tool for the identification of earthworm species and haplotypes.

BackgroundEarthworm communities are an important component of soil biodiversity and contribute to a number of ecosystem functions such as soil-nutrient cycling. Taxonomic identification is an essential requirement to assess earthworm biodiversity and functionality. Although morphological identification of species is labour-intensive, it is the most commonly used method due to a lack of cost-efficient alternatives. Molecular approaches to identify earthworms at species and haplotype level such as DNA barcoding are gaining popularity in science but are rarely applied in practice. In contrast to barcoding, the differentiation of PCR products based on their thermal denaturation properties using high-resolution melting (HRM) curve analysis is a fast and cost-efficient molecular closed-tube, post-PCR tool that allows identification of taxa.MethodsWe developed a HRM curve assay to identify eight earthworm species common to agricultural soils in Central Europe (Allolobophora chlorotica, Aporrectodea caliginosa, Apo. limicola, Apo. longa, Apo. rosea, Lumbricus castaneus, L. rubellus, and L. terrestris). For this, a new primer pair targeting a 158-bp long subregion of the cytochrome c oxidase I (COI) gene was designed. Our HRM assay was further tested for the differentiation of COI haplotypes using 28 individuals of the earthworm species Allo. chlorotica. Furthermore, we developed a novel extraction method for DNA from earthworm tissue that is fast and requires minimal consumables and laboratory equipment.ResultsThe developed HRM curve assay allowed identifying all eight earthworm species. Performing the assay on 28 individuals of the earthworm species Allo. chlorotica enabled the distinction among different COI haplotypes. Furthermore, we successfully developed a rapid, robust, scalable, and inexpensive method for the extraction of earthworm DNA from fresh or frozen tissue.ConclusionsHRM curve analysis of COI genes has the potential to identify earthworm species and haplotypes and could complement morphological identification, especially for juvenile or damaged individuals. Our rapid and inexpensive DNA extraction method from earthworm tissue helps to reduce the costs of molecular analyses and thereby promote their application in practice.

Development and validation of high-resolution melting assays for the detection of potentially virulent strains of Escherichia coli O103 and O121

Virulent strains of Shiga toxin-producing Escherichia coli (STEC) serogroups O103 and O121 are considered adulterants in beef. Two high-resolution melting (HRM) real-time PCR assays were standardized for the specific detection and discrimination of potentially virulent and avirulent strains of E. coli O103 and O121. The O103 HRM assay offered the possibility to distinguish clearly STEC O103:H2 from STEC O103:H25. The two standardized assays were extensively validated using 215 pure culture strains, laboratory inoculated food samples, and naturally contaminated beef (n = 84) and pork (n = 84) enrichments collected from the red meat surveillance program. Both HRM assays showed 100% inclusivity and exclusivity using pure culture strains and enriched spiked food samples. Data from this study shows the ability of the standardized assays to specifically detect the strains of each target serogroup and, most importantly, to differentiate the strains present into potentially virulent or avirulent groups. The assays standardized in this study can be helpful for food surveillance programs and help mitigate product loss due to the presence of avirulent strains lacking crucial virulence genes (stx and eae).

Detection and typing of dengue virus by one-step RT-PCR-based high-resolution melting assay.

Dengue is a rapidly spreading arboviral disease that can be transmitted through any of the four types of dengue virus (DENV) serotypes. Previous studies have observed that individuals who have a pre-existing secondary infection due to a different dengue serotype, experience severe forms of this disease. During a DENV outbreak, a time-sensitive preliminary diagnosis of the origin of DENV might be useful in controlling the epidemic. Here, we developed a rapid and accurate one-step TB Green RT-PCR-based high-resolution melting (HRM) assay to identify and serotype DENV using serotyping primers based on the alignment with the E gene. This assay had a detection limit of 7.7 × 102 (DENV 1), 3.8 × 102 (DENV 2), 6.2 × 102 (DENV 3), and 1.2 × 103 (DENV 4) RNA copies/mL. No cross-reactivity with the Chikungunya, Zika, and Japanese encephalitis viruses was observed. The feasibility of using this assay for clinical diagnosis was evaluated in DENV-positive patient sera. The HRM assay and the RT-qPCR had complete matched results derived from DENV detection, including 51 serum positive and 20 serum negative. Additionally, eight DENV 2 strains in the same serotype were successfully differentiated by an HRM assay. Thus, this assay facilitated accurate detection and serotyping of DENV, along with the time-sensitive identification of the infectious focus of different DENVs.

High-resolution Melting Analysis for NOTCH1 c.7541-7542delCT Mutation in Chronic Lymphocytic Leukemia: Prognostic Significance in Egyptian Patients.

The present study aimed to detect the prevalence of NOTCH1 c.7541-7542delCT mutation in Egyptian CLL patients using HRM assay and to assess its relation to patients' survival. The study included 50 newly diagnosed treatment-naïve CLL patients and 50 age and sex matched healthy controls. NOTCH1 c.7541-7542delCT mutation was detected using High-resolution melting (HRM) assay and direct Sanger sequencing. Outcome parameters included progression free survival (PFS) and overall survival (OS). NOTCH1 c.7541-7542delCT mutation was detected in 5 (10.0%) of CLL patients. No controls had NOTCH1 c.7541-7542delCT mutation. Similar results were obtained by direct Sanger sequencing yielding a sensitivity and specificity of 100.0% for HRM in detection of NOTCH1 c.7541-7542delCT mutation in the studied patients. In univariate analysis, predictors of OS included Trisomy 12, high LDH, presence of NOTCH1 c.7541-7542delCT mutation and lack of CR. In multivariate analysis, only lack of CR was found as a significant predictor of OS. HRM analysis is a sensitive method for detection of NOTCH1 c.7541-7542delCT mutation in CLL patients. This mutation may be linked to poor disease prognosis.

Development of a novel high resolution melting assay for identification and differentiation of all known 19 serovars of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory infectious disease responsible for global economic losses in the pig industry. From a monitoring perspective as well as due to the different courses of disease associated with the various serovars, it is essential to distinguish them in different herds or countries. In this study, we developed a novel high resolution melting (HRM) assay based on reference strains for each of the 19 known serovars and additional 15 clinical A. pleuropneumoniae isolates. The novel HRM comprises the species‐specific APP‐HRM1 and two serovar‐specific HRM assays (APP‐HRM2 and APP‐HRM3). APP‐HRM1 allowed polymerase chain reaction (PCR) amplification of apxIV resulting in an A. pleuropneumoniae specific melting curve, while nadV specific primers differentiated biovar 2 from biovar 1 isolates. Using APP‐HRM2 and APP‐HRM3, 13 A. pleuropneumoniae serovars can be determined by inspecting the assigned melting temperature. In contrast, serovar 3 and 14, serovar 9 and 11, and serovar 5 and 15 have partly overlapping melting temperatures and thus represent a challenge to accurately distinguish them. Consequently, to unambiguously ensure the correct assignment of the serovar, it is recommended to perform the serotyping HRM assay using a positive control for each serovar. This rapid and user‐friendly assay showed high sensitivity with 1.25 fg–125 pg of input DNA and a specificity of 100% to identify A. pleuropneumoniae. Characteristic melting patterns of amplicons might allow detecting new serovars. The novel HRM assay has the potential to be implemented in diagnostic laboratories for better surveillance of this pathogen.

Characterization and Aggressiveness of Take-All Root Rot Pathogens Isolated from Symptomatic Bermudagrass Putting Greens.

Take-all root rot is a disease of ultradwarf bermudagrass putting greens caused by Gaeumannomyces graminis (Gg), Gaeumannomyces sp. (Gx), Gaeumannomyces graminicola (Ggram), Candidacolonium cynodontis (Cc), and Magnaporthiopsis cynodontis (Mc). Many etiological and epidemiological components of this disease remain unknown. Improving pathogen identification and our understanding of the aggressiveness of these pathogens along with growth at different temperatures will advance our knowledge of disease development to optimize management strategies. Take-all root rot pathogens were isolated from symptomatic bermudagrass root and stolon pieces from 16 different golf courses. Isolates of Gg, Gx, Ggram, Cc, and Mc were used to inoculate 'Champion' bermudagrass in an in planta aggressiveness assay. Each pathogen was also evaluated at 10, 15, 20, 25, 30, and 35°C to determine growth temperature optima. Infected plant tissue was used to develop a real-time PCR high-resolution melt assay for pathogen detection. This assay was able to differentiate each pathogen directly from infected plant tissue using a single primer pair. In general, Ggram, Gg, and Gx were the most aggressive while Cc and Mc exhibited moderate aggressiveness. Pathogens were more aggressive when incubated at 30°C compared with 20°C. While they grew optimally between 24.4 and 27.8°C, pathogens exhibited limited growth at 35°C and no growth at 10°C. These data provide important information on this disease and its causal agents that may improve take-all root rot management.

3D reconstructed brain images reveal the possibility of the ogg1 gene to suppress the irradiation-induced apoptosis in embryonic brain in medaka (Oryzias latipes)

The accumulation of oxidative DNA lesions in neurons is associated with neurodegenerative disorders and diseases. Ogg1 (8-oxoG DNA glycosylase-1) is a primary repair enzyme to excise 7,8-dihydro-8-oxoguanine (8-oxoG), the most frequent mutagenic base lesion produced by oxidative DNA damage. We have developed ogg1-deficient medaka by screening with a high resolution melting (HRM) assay in Targeting-Induced Local Lesions In Genomes (TILLING) library. In this study, we identified that ogg1-deficient embryos have smaller brains than wild-type during the period of embryogenesis and larvae under normal conditions. To reveal the function of ogg1 when brain injury occurs during embryogenesis, we examined the induction of apoptosis in brains after exposure to gamma-rays with 10 Gy (137Cs, 7.3 Gy/min.) at 24 h post-irradiation both in wild-type and ogg1-deficient embryos. By acridine orange (AO) assay, clustered apoptosis in irradiated ogg1-deficient embryonic brains were distributed in a similar manner to those of irradiated wild-type embryos. To evaluate possible differences of gamma-ray induced apoptosis in both types of embryonic brains, we constructed 3D images of the whole brain based on serial histological sections. This analysis identified that the clustered apoptotic volume was about 3 times higher in brain of irradiated ogg1-deficient embryos (n = 3) compared to wild-type embryos (n = 3) (P = 0.04), suggesting that irradiation-induced apoptosis in medaka embryonic brain can be suppressed in the presence of functional ogg1. Collectively, reconstruction of 3D images can be a powerful approach to reveal slight differences in apoptosis induction post-irradiation.

A screening strategy for identifying the dominant variant of SARS-COV-2 in the fifth peak of Kurdistan- Iran population using HRM and Probe-based RT-PCR assay

By the emergence of SARS CoV-2 variants, many studies were developed to deal with it. The high transmissibility and mortality rate of some variants, in particular developing countries have caused the operation of simple diagnostic tests for genomic surveillance. In this study, we developed two assays of High Resolution Melting (HRM) and Probe-based RT-PCR as simple and inexpensive methods to identify the variants. We screened the mutations of del69–70, E484K, E484Q, D614G, L452R, and T478K in 100 cases from SARS-COV-2 positive patients in Kurdistan- Iran population. In general, the result of the two methods overlapped each other, nevertheless, we suggested HRM results be confirmed with a standard assay (Whole-Genome Sequencing). This work indicated that HRM as the rapid and inexpensive method could identify and categorize the variants of SARS CoV-2 and reduce the costs for carrying out sequencing.

A high-resolution melting assay to differentiate a peste des petits ruminants virus vaccine strain from field isolates in Turkey.

Peste des petits ruminants (PPR) mostly affects small ruminants. Vaccination with attenuated vaccines derived from PPR virus (PPRV) provides successful protection against the disease. However, current molecular methods are unable to distinguish vaccine strains from field strains. In this study, we used an EvaGreen-based high-resolution melting (HRM) assay to differentiate a PPRV vaccine strain (Nigeria 75/1) from Turkish field isolates of lineage IV based on melting peaks and melting profiles.