Melting Curve Research Articles

Implementation of a Real Time PCR protocol coupled with melting curve analysis for evaluating the clonality of the lymphocyte population in dogs

Genetic testing represents a major asset for discriminating between normal antigenic stimulation of B or T cells and malignant, pathological findings. The main difference between those two situations is the heterogenicity of lymphocyte populations as a result of diverse antigenic stimulation for the first situation, compared to single and identical population (clones) for the second situation. The present paper describes one of the many genetic assays capable of discriminating between normal and malignant, characterized by low time-to-result interval, accuracy, simplicity, and low cost comparing to other tests.

Application of real-time PCR-based multicolor melting curve with automatic analysis system in pregestational and prenatal thalassemia diagnoses.

To evaluate the value of the real-time PCR-based multicolor melting curve analysis (MMCA) with an automatic analysis system used in a mass thalassemia screening and prenatal diagnosis program. A total of 18,912 peripheral blood samples from 9456 couples and 1150 prenatal samples were detected by MMCA assay. All prenatal samples were also tested by a conventional method. Samples with unknown melting peaks, unusual peak height ratios between a wild allele and a mutant allele, or a discordant phenotype-genotype match were further studied by using multiplex ligation-dependent probe amplification (MLPA) or Sanger sequencing. All MMCA results were automatically analyzed and manually checked. The consistency between MMCA assay and conventional methods among prenatal samples was investigated. Except for initiation codon (T>G) (HBB:c.2T>G), all genotypes of thalassemia inside the scope of conventional methods were detected by MMCA assay. Additionally, 27 carriers with 10 rare HBB variants, 13 with α fusion gene, 1 with a rare deletion in α globin gene, and 1 with rare HBA variant were detected by using MMCA assay. MMCA can be an alternative approach used in routine thalassemia carrier screening and prenatal diagnosis for its high throughput, sufficient stability, low cost, and easy operation.

Thermal and spectroscopic profiles variation of cold-pressed raspberry seed oil studied by DSC, UV/VIS, and FTIR techniques

The characteristics of six cold-pressed raspberry seed oils, investigated by melting and crystallization phase transition analysis using differential scanning calorimetry (DSC), UV–VIS and Fourier transform infrared (FTIR) spectra, as well as the fatty acid composition, was the aim of this study. Two groups of oils samples were investigated: commercial (I-group) and laboratory-pressed (L-group). UV/VIS and FTIR spectra of raspberry seed oil revealed important wavelength and wavenumber ranges to indicate qualitative differences between I- and L-group. Regardless of the scanning rates, DSC melting curves were more suitable for detecting differences in raspberry seed oils than crystallization profiles. Based on the Principal component analysis of UV–VIS, FTIR and DSC data two groups of oils were distinguished. Fatty acids by GC analysis confirmed differences between oils, since the commercial oils exhibited a lower ratio of n-3/n-6 fatty acids than L-group oils. The obtained results provide practical information on the usefulness of DSC profiles and FTIR spectra for the comprehensive characterization of cold-pressed raspberry seed oils, especially in the field of B2B industries that need rapid and non-targeted instruments to confirm the authenticity of the purchased product.

A-268 Evaluation of a Real-Time Multiplex PCR Assay for Simultaneous Detection of Chlamydia trachomatis, Neisseria gonorrhoeae, and Trichomonas vaginalis in Urine Specimens

Abstract Background Infections with Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV) are among the most prevalent and treatable sexually transmitted diseases in the US. Currently, most available testing methods require patient samples to be split between CT/NG and TV assays. Here, we evaluate performance characteristics of the Seegene Novaplex STI Essential Assay, a real-time multiplex polymerase chain reaction assay for concurrent amplification and detection of multiple pathogens including CT, NG, and TV. Methods To prepare urine samples for extraction, 1 mL of each sample was centrifuged at 13 000 rpm for 15 min. The supernatant was discarded and the pellet was rehydrated in 190 µL of saline. Nucleic acid extraction was performed on a CyBio® FeliX liquid handler (Analytik Jena, Germany) using MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, MA). PCR amplification and detection were performed using Seegene Novaplex STI assay (Seegene Inc, South Korea) on a CFX96™ System (Bio-Rad Laboratories, CA). The assay utilizes Seegene MuDT™ technology that allows detection of multiple targets in a single channel without melting curve analysis. For method validation, precision studies were performed within-run and over 5 days. Limits of detection were verified by spiking known negative urine samples with CT/NG standards from Exact Diagnostics (Bio-Rad Laboratories, CA) and TV standards from ZeptoMetrix® (Antylia Scientific, IL). Method comparison was carried out by extracting and analyzing split patient urine samples against cobas® CT/NG assay (Roche Diagnostics, IL) and Solana® Trichomonas assay (Quidel, CA) performed by a reference laboratory. Additional accuracy studies were performed by spiking 20 negative patient samples with variable concentrations of the three pathogens. Results Within-run and between-day precision studies demonstrated consistent and reproducible results for all pathogens. Limit of detection was established at 10 copies/mL for CT and NG and 10 cells/mL for TV. Results of the spike-and-recovery studies were consistent with expected results for all three targets across evaluated concentrations. Patient correlation studies demonstrated 100% agreement with results obtained by the reference laboratory for both NG (32 total samples, 11 positive) and TV (22 total samples, 4 positive), and 96% agreement for CT (29 total samples, 10 positive). Conclusion The Seegene Novaplex STI Essential Assay offers a rapid and reliable method for detecting CT, NG, and TV in urine samples. The capability of the assay to detect multiple targets in a single sample well improves efficiency and turnaround time.

Synthesis and Biological Activity of a New Indenoisoquinoline Copper Derivative as a Topoisomerase I Inhibitor.

Topoisomerases are interesting targets in cancer chemotherapy. Here, we describe the design and synthesis of a novel copper(II) indenoisoquinoline complex, WN198. The new organometallic compound exhibits a cytotoxic effect on five adenocarcinoma cell lines (MCF-7, MDA-MB-231, HeLa, HT-29, and DU-145) with the lowest IC50 (0.37 ± 0.04 μM) for the triple-negative MDA-MB-231 breast cancer cell line. Below 5 µM, WN198 was ineffective on non-tumorigenic epithelial breast MCF-10A cells and Xenopus oocyte G2/M transition or embryonic development. Moreover, cancer cell lines showed autophagy markers including Beclin-1 accumulation and LC3-II formation. The DNA interaction of this new compound was evaluated and the dose-dependent topoisomerase I activity starting at 1 μM was confirmed using in vitro tests and has intercalation properties into DNA shown by melting curves and fluorescence measurements. Molecular modeling showed that the main interaction occurs with the aromatic ring but copper stabilizes the molecule before binding and so can putatively increase the potency as well. In this way, copper-derived indenoisoquinoline topoisomerase I inhibitor WN198 is a promising antitumorigenic agent for the development of future DNA-damaging treatments.

A rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for detection of Entamoeba histolytica and Giardia duodenalis.

We developed a rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for two common intestinal parasites-Entamoeba histolytica and Giardia duodenalis, where early detection may be helpful. The mLAMP assay was optimized for detection of DNA of E. histolytica (18S rRNA gene) and G. duodenalis (Elongation factor 1 alfa gene) from standard strains by using six specific primers (FIP, BIP, F3, B3, FLP, BLP) for each gene target. The amplification time was 16-26 min for E. histolytica and 10-15 min for G. duodenalis, and the parasites could be distinguished based on melting curve analysis for specific annealing temperatures (Tm) of 84-86°C and 88-90°C for E. histolytica and G. duodenalis, respectively. The analytical sensitivity was one fg, and no cross-reactivity with other intestinal pathogens was observed. Thus, the mLAMP assay could detect and clearly distinguish E. histolytica and G. duodenalis with a rapid turnaround time and excellent analytical sensitivity and specificity.

Machine-Learning Accelerated First-Principles Accurate Modeling of the Solid-Liquid Phase Transition in MgO under Mantle Conditions.

While accurate measurements of MgO under extreme high-pressure conditions are needed to understand and model planetary behavior, these studies are challenging from both experimental and computational modeling perspectives. Herein, we accelerate density functional theory (DFT) accurate calculations using deep neural network potentials (DNPs) trained over multiple phases and study the melting behavior of MgO via the two-phase coexistence (TPC) approach at 0-300 GPa and ≤9600 K. The resulting DNP-TPC melting curve is in excellent agreement with existing experimental studies. We show that the mitigation of finite-size effects that typically skew the predicted melting temperatures in DFT-TPC simulations in excess of several hundred kelvin requires models with ∼16 000 atoms and >100 ps molecular dynamics trajectories. In addition, the DNP can successfully describe MgO metallization well at increased pressures that are captured by DFT but missed by classical interatomic potentials.

Advances in RNA Labeling with Trifluoromethyl Groups.

Fluorine labeling of ribonucleic acids (RNA) in conjunction with 19 F NMR spectroscopy has emerged as a powerful strategy for spectroscopic analysis of RNA structure and dynamics, and RNA-ligand interactions. This study presents the first syntheses of 2'-OCF3 guanosine and uridine phosphoramidites, their incorporation into oligoribonucleotides by solid-phase synthesis and a comprehensive study of their properties. NMR spectroscopic analysis showed that the 2'-OCF3 modification is associated with preferential C2'-endo conformation of the U and G ribose in single-stranded RNA. When paired to the complementary strand, slight destabilization of the duplex caused by the modification was revealed by UV melting curve analysis. Moreover, the power of the 2'-OCF3 label for NMR spectroscopy is demonstrated by dissecting RNA pseudoknot folding and its binding to a small molecule. Furthermore, the 2'-OCF3 modification has potential for applications in therapeutic oligonucleotides. To this end, three 2'-OCF3 modified siRNAs were tested in silencing of the BASP1 gene which indicated enhanced performance for one of them. Importantly, together with earlier work, the present study completes the set of 2'-OCF3 nucleoside phosphoramidites to all four standard nucleobases (A, U, C, G) and hence enables applications that utilize the favorable properties of the 2'-OCF3 group without any restrictions in placing the modification into the RNA target sequence.

Semiautomated Electrochemical Melting Curve Analysis Device for the Detection of an Osteoporosis Associated Single Nucleotide Polymorphism in Blood.

The detection of single nucleotide polymorphisms (SNPs) is of increasing importance in many areas including clinical diagnostics, patient stratification for pharmacogenomics, and advanced forensic analysis. In the work reported, we apply a semiautomated system for solid-phase electrochemical melting curve analysis (éMCA) for the identification of the allele present at a specific SNP site associated with an increased risk of bone fracture and predisposition to osteoporosis. Asymmetric isothermal recombinase polymerase amplification using ferrocene labeled forward primers was employed to generate single stranded redox labeled amplicons. In a first approach to demonstrate the proof of concept of combining asymmetric RPA with solid-phase éMCA, a simplified system housing a multielectrode array within a polymeric microsystem, sandwiched between two aluminum plates of a heater device, was used. Sample manipulation through the microfluidic channel was controlled by a syringe pump, and an external Ag/AgCl reference electrode was employed. Individual electrodes of the array were functionalized with four different oligonucleotide probes, each probe equivalent in design with the exception of the middle nucleotide. The isothermally generated amplicons were allowed to hybridize to the surface-tethered probes and subsequently subjected to a controlled temperature ramp, and the melting of the duplex was monitored electrochemically. A clear difference between the fully complementary and a single mismatch was observed. Having demonstrated the proof-of-concept, a device for automated éMCA with increased flexibility to house diverse electrode arrays with internal quasi-gold reference electrodes, higher resolution, and broader melting temperature range was developed and exploited for the detection of SNP hetero/homozygosity. Using the optimized conditions, the system was applied to the identification of the allele present at an osteoporosis associated SNP site, rs2741856, in 10 real fingerprick/venous blood samples, with results validated using Sanger sequencing.

Comparison of the sensitivity of restriction analysis and PCR with high-resolution melting curve analysis for the detection of the R882H mutation in the DNMT3A gene

Introduction. DNMT3A is one of the most frequently mutated genes in acute myeloid leukemia (AML), and the R882H (G>A) substitution is one of the most common mutations in this gene. Restriction analysis and high resolution melting (HRM) analysis are the most common methods used to detect this mutation, but the sensitivity of these approaches varies greatly from study to study. Purpose of the study. Determine the sensitivity of commonly used methods for assessing the R882H mutation content of a sample under controlled conditions. Methods. We compared the sensitivity of restriction analysis and HRM on presynthesized DNA samples with varying levels of mutant template in the sample and provided detailed protocols for reproducing our results by other researchers. Results. We found that the detection limit of the R882H mutation in the DNMT3A gene was 20% for restriction analysis and 40% for HRM. Conclusion. The results obtained in this work are important for identifying restriction analysis and HRM as suitable methods for use in laboratory diagnosis of the R882H mutation for patients with AML with a high mutational load.

Visual single nucleotide polymorphism (SNP) detection for ALDH2 genotyping based on multiplex ligation probe amplification (MLPA) and lateral flow assay

Current techniques for single nucleotide polymorphism (SNP) detection require laborious experimental procedures, expensive and sophisticated instruments, as well as complex data analysis, which make it difficult to meet the demand for routine clinical laboratories or point of care testing (POCT). In this study, the authors propose a reliable and cost-effective method by integrating multiplex ligation probe amplification (MLPA) and gold nanoparticles (AuNPs)-based lateral flow assay (LFA) for visual detection of the ALDH2 gene rs671(G > A) polymorphism. The genotyping result can be easily observed with the naked eye within 5–10 min after loading the MLPA products on the LFA device. This system demonstrates high sensitivity with LOD down to 1 ng, meeting the requirement in clinical diagnostic. Furthermore, this method has been implemented in clinical practices to identify the genotypes of the ALDH2 gene. The results obtained from the MLPA-LFA, Sanger sequencing, and high-resolution melting (HRM) curve analysis demonstrate a perfect agreement rate of 100%. Compared to conventional genotyping assays, this system exhibits the following advantages, including visual interpretation of results, minimal requirements for sophisticated instruments and highly skilled technicians, time efficiency, as well as low cost with a price approximately at 5 dollars. We believe that the MLPA-LFA system is a robust and versatile method with the potential to detect other SNPs related to disease susceptibility and risk, as well as drug metabolism and therapeutic effects.

Genetic Variability in Vitamin D Receptor and Migraine Susceptibility: A Southeastern European Case-Control Study.

Migraine is a common primary headache disorder with both environmental and genetic inputs. Cumulative evidence indicates an association between vitamin D and headache. Unravelling the precise role of vitamin D and its receptor in the pathophysiology of migraine can eventually contribute to more efficient prevention and management of this headache disorder. The aim of the study was to investigate the relation of the three most studied VDR variants, i.e., FokI (rs2228570), TaqI (rs731236) and BsmI (rs1544410), with migraine susceptibility and distinct clinical phenotypes in a Southeastern European case-control population residing in Greece. DNA was extracted from 191 unrelated patients diagnosed with migraine and 265 headache-free controls and genotyped using real-time PCR (LightSNiP assays) followed by melting curve analysis. Genotype frequency distribution analysis of the TaqI and BsmI variants showed a statistically significant difference between migraine cases and controls. In addition, subgroup analyses revealed a significant association between all three studied VDR variants, particularly with a migraine without aura subtype. Therefore, the current study provides supporting evidence for a possible association of VDR variants with migraines, particularly migraine without aura susceptibility in Southeastern Europeans residing in Greece, further reinforcing the emerging role of vitamin D and its receptor in migraines.

Passive Surveillance of SARS-CoV-2 in Adult Blacklegged Ticks (Ixodes scapularis) from Northeast Pennsylvania.

Monitoring the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wildlife is vital to public health. White-tailed deer (Odocoileus virginianus) in the United States have tested positive for SARS-CoV-2, and their interactions with blacklegged ticks (Ixodes scapularis) raise the question of whether or not these ticks also carry SARS-CoV-2. In this study, 449 blacklegged ticks from Northeast Pennsylvania were collected in the fall of 2022 and tested via RT-qPCR for the presence of SARS-CoV-2. Fourteen ticks were amplified with late quantification cycles (Cq) using primers from two nucleocapsid genes (N1 and N2) via TaqMan assays. Three of these samples were amplified on a SYBR green assay during secondary testing. However, melt curve and gel electrophoresis analysis verified negative results for these three samples. Genetic sequencing was performed on one of the three samples to look for potential cross-reactions causing the amplification observed. However, no significant match was found in the NCBI database. Although all 449 blacklegged ticks were negative for SARS-CoV-2, I. scapularis should continue to be tested for COVID-19. If blacklegged ticks test positive for COVID-19 in the future, research should focus on determining the stability of SARS-CoV-2 with the tick vector and the potential for transmission through tick bites.

Large-area Fe–C eutectic fixed-points for radiation and contact thermometry

Abstract High-quality metal (carbide)–carbon eutectic materials based on high-temperature fixed points (HTFPs) are widely used in radiometry and thermometry as reference standards. HTFPs on the base of iron–carbon (Fe–C) binary eutectic alloys, with a nominal melting temperature of about 1154 °C (just above the copper freezing point of 1084.62 °C), are one of the promising candidates among the eutectic materials. To establish new HTFPs as reference metrological tools for high-temperature thermometry, their performance should be thoroughly investigated regarding reproducibility and stability. In this work, two large-area (8 mm aperture, 107 mm cavity/thermowell length) Fe–C fixed-point cells were constructed and studied in detail using a radiation thermometer and two different thermocouples (TCs). Three different furnaces were used to explore the thermal behaviors of the cells at various furnace gradients and furnace offsets. The melting temperature at the inflections point of the melting curves of the cells studied across extensive measurement campaigns demonstrated good performance with repeatability of less than 9 mK (assessed from four successive runs) and reproducibility—less than 100 mK (at different furnaces and furnace offsets). The melting temperature agreement between both cells in the same experimental conditions was better than 30 mK. In addition, the equivalence of the developed large-area cells and a small-area radiometric cell (3 mm cavity aperture, and 35 mm cavity length) were comparatively examined in the same experimental conditions. The coherence of the obtained results for the melting temperature of large-area Fe–C cells indicates the feasibility of using large-volume cells for precise calibration of both radiation thermometers and TCs.

Shock-induced melting and crystallization in titanium irradiated by ultrashort laser pulse

Modification of titanium microstructure after propagation of a melting shock wave (SW) generated by a femtosecond laser pulse is investigated experimentally and analyzed using hydrodynamic and atomistic simulations. Scanning and transmission electron microscopy with analysis of microdiffraction is used to determine the microstructure of modified subsurface layers of titanium. We found that two layers are modified beneath the surface. A top surface polycrystalline layer of nanoscale grains is formed from shock-molten material via rapid crystallization. In a deeper subsurface layer, where the shock-induced melting changes into plastic deformation due to attenuation of SW, the grain structure of solid is considerably affected, which results in a grain size distribution differing from that in the intact titanium. Molecular dynamics simulation of single-crystal titanium reveals that the SW front continues to melt even after its temperature drops below the melting curve Tm(P). The enormous shear stress of ∼12 GPa generated in a narrow SW front leads to free slip of atomic planes, collapse of the crystal lattice, and formation of a supercooled metastable melt. Such melt crystallizes in an unloading tail of SW. The mechanical melting ceases after drop in the shear stress giving rise to the shock-induced plastic deformation. The last process triggers a long-term rearrangement of atomic structures in solid. The overall depth of modified layers is limited by SW attenuation to the Hugoniot elastic limit and can reach several micrometers. The obtained results reveal the basic physical mechanisms of surface hardening of metals by ultrashort laser pulses.

Occurrence, Characteristics, and qPCR-Based Identification of Pectobacterium versatile Causing Soft Rot of Chinese Cabbage in China.

Pectobacterium is one of the most important genera of phytopathogenic bacteria. It can cause soft-rot diseases on a wide range of plant species across the world. In this study, three Pectobacterium strains (KC01, KC02, and KC03) were isolated from soft-rotted Chinese cabbage in Beijing, China. These three strains were identified as Pectobacterium versatile based on phylogenetic analysis of Pectobacterium 16S ribosomal RNA, pmrA, and 504 Pectobacterium core genes, as well as a genomic average nucleotide identity analysis. Their biochemical characteristics were found to be similar to the P. versatile type strain ICMP9168T but differed in response to citric acid, stachyose, D-glucuronic acid, dextrin, and N-acetyl-β-D-mannosamine. All of the tested P. versatile strains showed different carbohydrate utilization abilities compared with P. carotovorum and P. odoriferum, particularly in their ability to utilize D-arabitol, L-rhamnose, and L-serine. Under laboratory conditions, the maceration ability of P. versatile on Chinese cabbage was the highest at 28°C, compared with those at 13, 28, 23, and 33°C. Additionally, P. versatile could infect all of the 17 known Pectobacterium host plants, except for Welsh onion (Allium fistulosum). A SYBR Green quantitative PCR (qPCR) detection system was developed to distinguish P. versatile from other soft-rot bacteria based on the combined performance of melting curve (with a single melting peak at around 85°C) and fluorescence curve (with cycle threshold <30) when the bacterial genomic DNA concentration was in the range of 10 pg/µl to 10 ng/µl. This study is the first to report the presence of P. versatile on Chinese cabbage in China, as well as a specific and sensitive qPCR assay that can be used to quickly identify P. versatile. The work contributes to a better understanding of P. versatile and will facilitate the effective diagnosis of soft-rot disease, ultimately benefitting commercial crop production.

An extraction-free method for rapid detection of CYP2C19 * 2/3/17 polymorphisms in one tube using melting curve analysis.

Drug-metabolizing enzymes play an important role in the metabolism of drugs in vivo. Their activity is an important factor affecting the rate of drug metabolism, which directly determines the intensity and persistence of drug action. Patients taking medication can be divided into different metabolic types through detection of CYP2C19 drug-metabolizing enzyme gene polymorphisms, which can then be used for medication guidance for clopidogrel. Here, we describe a detection method based on real-time polymerase chain reaction. This method uses multicolor melting curve analysis to accurately identify different mutation sites and genotypes of CYP2C19 * 2, CYP2C19 * 3, and CYP2C19 * 17. The detection limit of plasmid samples was 1 copies/μl; that of genomic samples was 0.1ng/μl. The system can detect nine types of CYP2C19 * 2/3/17 at three sites in one tube, quickly achieving detection within 1h. Combined with the sample release agent, sample extraction was completed in 5 s, achieving rapid diagnosis without extraction for timely diagnosis and treatment. Furthermore, the system is not limited to blood samples and can also be applied to oropharyngeal and saliva samples, increasing sampling diversity and convenience. When using clinical blood samples (n=93), the detection system we established was able to quickly and accurately identify different genotypes, and the accuracy and effectiveness of the detection were confirmed by Sanger sequencing. Due to its accuracy, rapidity, simple operation and low cost, detection technology based on real-time polymerase amplification combined with melting curve analysis is expected to become a powerful tool for detecting and guiding clopidogrel use in countries with limited resources. This article is protected by copyright. All rights reserved.

Discrimination of SARS-CoV-2 omicron variant and its lineages by rapid detection of immune-escape mutations in spike protein RBD using asymmetric PCR-based melting curve analysis

BackgroundThe SARS-CoV-2 Omicron strain has multiple immune-escape mutations in the spike protein receptor-binding domain (RBD). Rapid detection of these mutations to identify Omicron and its lineages is essential for guiding public health strategies and patient treatments. We developed a two-tube, four-color assay employing asymmetric polymerase chain reaction (PCR)-based melting curve analysis to detect Omicron mutations and discriminate the BA.1, BA.2, BA.4/5, and BA.2.75 lineages.MethodsThe presented technique involves combinatory analysis of the detection of six fluorescent probes targeting the immune-escape mutations L452R, N460K, E484A, F486V, Q493R, Q498R, and Y505H within one amplicon in the spike RBD and probes targeting the ORF1ab and N genes. After protocol optimization, the analytical performance of the technique was evaluated using plasmid templates. Sensitivity was assessed based on the limit of detection (LOD), and reliability was assessed by calculating the intra- and inter-run precision of melting temperatures (Tms). Specificity was assessed using pseudotyped lentivirus of common human respiratory pathogens and human genomic DNA. The assay was used to analyze 40 SARS-CoV-2–positive clinical samples (including 36 BA.2 and 4 BA.4/5 samples) and pseudotyped lentiviruses of wild-type and BA.1 viral RNA control materials, as well as 20 SARS-CoV-2–negative clinical samples, and its accuracy was evaluated by comparing the results with those of sequencing.ResultsAll genotypes were sensitively identified using the developed method with a LOD of 39.1 copies per reaction. The intra- and inter-run coefficients of variation for the Tms were ≤ 0.69% and ≤ 0.84%, with standard deviations ≤ 0.38 °C and ≤ 0.41 °C, respectively. Validation of the assay using known SARS-CoV-2–positive samples demonstrated its ability to correctly identify the targeted mutations and preliminarily characterize the Omicron lineages.ConclusionThe developed assay can provide accurate, reliable, rapid, simple and low-cost detection of the immune-escape mutations located in the spike RBD to detect the Omicron variant and discriminate its lineages, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.

Effects of Dietary Supplements of DHA-enriched Micro Algae Diet on Physical and Technological Properties of Dairy Cow Milk Fat

This study examined the effect of dietary supplementation of DHA-enriched micro algae diet on physical and technological properties of dairy milk fat in terms of the dynamic crystallization and melting behaviour. Two dairy cows were subjected to feeding regime of DHA-enriched micro algae diet and control diet. The experiment was carried out during 21-d to determine the normal (control) and DHA-enriched micro algae diet modified milk fat that were taken for further analysis. The melting and crystallization behaviour of the milk fat from the cows fed control and DHA-enriched micro algae diets was studied using differential scanning calorimetry (DSC), Q1000 (TA Instruments, New Castle, DE, United States). DHA-enriched micro algae supplementation strongly affected the melting and crystallization properties of milk fat. Generally, the onset temperature (°C) of milk fat crystallization was significantly lower in DHA-enriched milk fat as compared to the control. The quantity of heat released by fat crystallization expressed as J/g (peak area) was significantly lower in enriched milk fat. DHA-enriched milk fat also had a lower peak maximum temperature as compared to control in all samples investigated. All melting curves displayed two peaks (lower melting and higher melting peaks) and for melting peaks, DHA-enriched milk fat melted at significantly lower temperature as compared to the control indicating an increase in the degree of unsaturation of milk fat. Melting offset temperature was significantly lower for DHA-enriched milk fat as compared to the control. It can be concluded that from the results of this study, micro algae supplementation significantly altered the milk fat composition and positively affected melting and crystallization behaviour of milk fat.

Quantitative Investigation of Methylation Heterogeneity by Digital Melting Curve Analysis on a SlipChip for Atrial Fibrillation.

Methylation is an essential epigenetic modification involved in regulating gene expression and maintaining genome stability. Methylation patterns can be heterogeneous, exhibiting variations in both level and density. However, current methods of methylation analysis, including sequencing, methylation-specific PCR, and high-resolution melting curve analysis (HRM), face limitations of high cost, time-consuming workflows, and the difficulty of both accurate heterogeneity analysis and precise quantification. Here, a droplet array SlipChip-based (da-SlipChip-based) digital melting curve analysis (MCA) method was developed for the accurate quantification of both methylation level (ratio of methylated molecules to total molecules) and methylation density (ratio of methylated CpG sites to total CpG sites). The SlipChip-based digital MCA system supplements an in situ thermal cycler with a fluorescence imaging module for real-time MCA. The da-SlipChip can generate 10,656 droplets of 1 nL each, which can be separated into four lanes, enabling the simultaneous analysis of four samples. This method's clinical application was demonstrated by analyzing samples from ten healthy individuals and twenty patients with atrial fibrillation (AF), the most common arrhythmia. This method can distinguish healthy individuals from those with AF of both the paroxysmal and persistent types. It also holds potential for broader application in various research and clinical settings requiring methylation analysis.