Melting Curve Research Articles

Real-time fluorescence growth curves for viable bacteria quantification in foods

Here, for the first time, we used a membrane permeable fluorescent nucleic acid stain (SYBR Green) to trace the in-vivo DNA replication during bacterial binary fission. Such stain did not influence the growth of bacteria. Nor did the bacteria degrade the stain, enabling the fluorescent microplate reader to monitor sensitively the growth of the bacteria. Hence, a real-time fluorescence growth curve (RTFGC) method was put forward for the sensitive quantification of viable bacteria in foods. Using E. coli O157:H7 as a bacteria model, the RTFGC method could quantify bacteria within the range of 10 to 1 × 106 cfu/mL, with a linear correlation coefficient R2 of 0.997. It was found that melting curve was unique for a particular bacterial strain, which could be used for contamination identifications. Good practicability of the RTFGC in quantifying E. coli O157:H7 from tap water, juices, and milks was demonstrated.

High Pressure Melting Curve of Fe-Si: Implication for the Thermal Properties in Mercury's Core.

The motion of liquid iron (Fe) alloy materials in the outer core drives the dynamo, which generates Mercury's magnetic field. The assessment of core models requires laboratory measurements of the melting temperature of Fe alloys at high pressure. Here, we experimentally determined the melting curve of Fe9wt%Si and Fe17wt%Si up to 17GPa using in situ and ex situ measurements of intermetallic fast diffusion that serves as the melting criterion in a large-volume press. Our determined melting slopes are comparable with previous studies up to about 17GPa. However, when extrapolated, our melting slopes significantly deviate from previous studies at higher pressures. For Mercury's core with a model composition of Fe9wt%Si, the melting temperature-depth profile determined in our study is lower by ∼150-250K when compared with theoretical calculations. Using the new melting curve of Fe9wt%Si and the electrical resistivity values from a previous study of Fe8.5wt%Si, we estimate that the electronic thermal conductivity of liquid Fe9wt%Si is 30 Wm-1K-1 at the Mercury's CMB pressure of 5GPa and 37Wm-1K-1 at an assumed ICB of 21GPa, corresponding to heat flux values of 23mWm-2 and 32mWm-2, respectively. These values provide new constraints on the core models.

Detection and differentiation of fowl adenovirus serotype 4 and duck adenovirus 3 using high resolution melting curve assay

Fowl adenovirus type 4 (FAdV-4) and duck adenovirus type 3 (DAdV-3) are the causative agents of clinical diseases in poultry and have caused considerable economic losses to the waterfowl industry in China. Both FAdV-4 and DAdV-3 are classified into the genus Aviadenovirus under the family Adenoviridae. The high-resolution melting (HRM) assay has become a useful method for virus genotyping, which offers the possibility of rapidly developing a differentiation technique in which the melting profile depends on the GC content of the product in the qPCR platform. The aim of this study was to develop a qPCR-HRM assay for sensitive FAdV-4 and DAdV-3 detection and differentiation. Here, specific primers were designed on the basis of the 100 K genes of FAdV-4 and DAdV-3, and a qPCR-HRM assay was established through optimization of the reaction conditions. A specificity test revealed that this method could detect only FAdV-4 and DAdV-3, with no cross-reaction with other common duck-derived viruses. A sensitivity test revealed that the lowest detection limits of FAdV-4 and DAdV-3 were 2.84 copies/µL and 2.85 copies/µL, respectively. A repeatability test demonstrated that the coefficient of variation was less than 2.5 % in both the intragroup and the intergroup analyses. Field sample distributions of FAdV-4 and DAdV-3 were investigated, and the percentages of DAdV-3-positive, FAdV-4-positive and coinfection-positive in Muscovy ducks were 27.78 %, 16.67 % and 11.11 %, respectively. Further studies are needed to provide more insight into the pathogenesis of FAdV-4 and DAdV-3 coinfection in ducks. In conclusion, the qPCR-HRM assay provides an accurate, sensitive, reliable and cost-effective alternative method for detecting and distinguishing FAdV-4 and DAdV-3.

Newborn screening for deafness genes with cord blood-based multicolour melting curve analysis

Newborn screening for deafness genes with cord blood-based multicolour melting curve analysis

The disordered negatively charged C-terminus of the large HECT E3 ubiquitin ligase HERC2 provides structural and thermal stability to the HECT C-lobe.

Homologous to the C-terminus of E6AP (HECT) and RCC1-like domain (RLD)-containing protein 2 (HERC2) is a large, 528 kDa E3 ubiquitin ligase that is associated with cancer, oculocutaneous albanism type 2, Prader-Willi syndrome, and other neurological diseases. HERC2 has been found to contribute to double-stranded DNA break repairs, tumor suppression, maintaining centrosome architecture, and ubiquitylation. The C-terminal portion of the HECT domain (C-lobe) of HERC2 is responsible for transferring ubiquitin to a substrate but the precise function of the other eight domains in HERC2 are unknown. Interestingly, HERC2 contains a unique and negatively charged C-terminal tail adjoined to the C-lobe that is predicted to act as a linker to promote interactions between HERC2 and its binding partners. This study aims to better understand the function and relevance of HERC2 in disease by investigating the structural aspects of the HERC2 C-lobe and HERC2 C-terminal tail using AlphaFold followed by molecular dynamics (MD) simulations, multidimensional nuclear magnetic resonance (NMR), and circular dichroism (CD). Secondary structure content analysis from MD simulations and the fully resonance assigned 1H-15N HSQC spectra of the HERC2 C-lobe and the isolated C-terminal tail confirm that the C-lobe is well-folded but the C-terminal tail is disordered. CD melting curves indicate that the flexible C-terminal tail provides improved stability to the C-lobe. Additionally, MD simulations have identified that the interaction between residues D4829 and R4728 is prevalent among the non-bonded contacts between the tail and the C-lobe. Overall, our results demonstrate that the negatively charged C-terminal tail is disordered, provides stability to the C-lobe, and may act as a flexible scaffold for protein-protein interactions.

A note on the direction of core solidification in asteroids, the iron melting curve, and phase equilibria parameterizations

The accuracy of the pressure/temperature/composition parameterization of Buono and Walker (2011) to describe the liquidus of iron and the Fe-FeS system is examined. In the pressure range critical for asteroidal core crystallization (0- ∼2 GPa), the model predicts a shape for the iron melting curve (initially negatively sloped, and turning over near 0.7 GPa) that is inconsistent with previous experimental observations, thermodynamic constraints, and millennia of empirical metallurgical observations. Dodds et al. (2025) recently used this model to derive notable conclusions about the behavior of the solidifying cores of asteroids: the robustness of their conclusions is assessed. Two basic caveat emptor guidelines for employing parameterizations of phase equilibria data are suggested: (1) ensure that the model's fit is consistent with simple thermodynamic expectations; and (2) verify that the data used to formulate the model provide adequate coverage in the region of interest.

Rapid detection of the SARS-CoV-2 omicron variants based on high-resolution melting curve analysis

With the continuous spread of the SARS-CoV-2 globally, viral mutations have accumulated. As a result, SARS-CoV-2 became more contagious, and has a higher risk of immune escape and reinfection. To identify variants and have an awareness of the prevalence of these variants, this study selected four segments containing mutations on the S gene of the SARS-CoV-2. Then a rapid and convenient variants detection method was established using high-resolution melting(HRM) analysis combined with nested polymerase chain reaction(PCR). The total detection process takes about 5 h. Through comprehensive analysis of the results from the four reaction systems, the identification of seven important Omicron variants(BA.2, BA.2.75, BA.5.2, BF.7, BQ.1, XBB.1 and XBB.2) can be achieved, with significant differentiation in the melting curves of each variant group. The method established in this study was used to genotype positive specimens in COVID-19 nucleic acid testing, the overall concordance rate compared to whole genome sequencing results was 88.9%, and the positive concordance rate of each sublineage was greater than 80% and the negative concordance rate was greater than 94.4%. The detection of clinical specimens has demonstrated that the HRM analysis established in this study is an effective, rapid and convenient variant identification method, which can be used for monitoring SARS-CoV-2 variants and has important value in addressing public health issues caused by the ongoing mutations of the SARS-CoV-2.

Upregulation of interferon gamma gene expression among asymptomatic malaria adults in Bayelsa State, Nigeria

Despite concerted efforts, malaria remains a global public health problem. Treatment for asymptomatic malaria will reduce the health care burden of malaria. However evidence-based data for the presence of systemic pathology in asymptomatic malaria, which might inform treatment remains limited. The present study investigated the current prevalence of asymptomatic malaria in Bayelsa State, Nigeria and differences in interferon gamma (IFNγ) mRNA expression between asymptomatic malaria individuals (AMIs) and uninfected individuals (UIs), in relation to ABO blood groups and hemoglobin genotype (Hb) variants. A cross-sectional design was employed to randomly select 146 non-febrile participants from Sagbama LGA, Bayelsa State. Plasmodium falciparum infection was determined using high-resolution melting analysis and participants were grouped into UIs and AMIs. Hb, blood groups, and IFNγ mRNA expression were determined using electrophoresis, agglutination, and real-time PCR method respectively. Logistic regression and t-test were used to determine significant differences between groups at p < 0.05. The current prevalence of asymptomatic malaria in Bayelsa State was found to be 89.73 %. Age and sex were associated with asymptomatic malaria (p < 0.05). Plasmodium falciparum DNA melt curves for AMIs aligned at 83.89 ± 0.34 °C, while the amplification cycle thresholds were below 30 cycles. Compared with UIs, the gene expression of IFNγ mRNA was significantly (p < 0.001) higher among AMIs. Also, among AMIs those with A, B, and AB blood groups expressed significantly higher levels of IFNγ mRNA compared with blood groups O AMIs. Furthermore, AMIs with HbAA expressed significantly higher levels of IFNγ mRNA compared with HbAS AMIs. The novelty of the present study is the demonstration of the existence of systemic pathology in asymptomatic malaria and the demonstration that the intensity of systemic pathology is dependent on blood types and haemoglobin genotype variants. Findings of the present study have implications for policy creation towards asymptomatic malaria treatment.

Development of a whole-cell SELEX process to select species-specific aptamers against Aspergillus niger

BackgroundSpores produced by the filamentous fungus Aspergillus niger are abundant in a variety of environments. The proliferation of this fungus in indoor environments has been associated to health risks and its conidia can cause allergic reaction and severe invasive disease in animals and humans. Therefore, the detection and monitoring of Aspergillus conidia is of utmost importance to prevent serious fungal infections and contaminations. Among others, aptamers could serve as biosensors for the specific detection of fungal spores.ResultsIn this study, DNA aptamers specific to conidia of A. niger were developed by optimizing a whole-cell SELEX approach. Three whole-cells SELEX experiments were performed in parallel with similar conditions. Quantification of recovered ssDNA and melting curve analyses were applied to monitor the ongoing SELEX process. Next-generation sequencing was performed on selected recovered ssDNA pools, allowing the identification of DNA aptamers which bind with high affinity to the target cells. The developed aptamers were shown to be species-specific, being able to bind to A. niger but not to A. tubingensis or to A. nidulans. The binding affinity of two aptamers (AN01-R9-006 and AN02-R9-185) was measured to be 58.97 nM and 138.71 nM, respectively, which is in the range of previously developed aptamers.ConclusionsThis study demonstrates that species-specific aptamers can be successfully developed via whole-cell SELEX to distinguish different Aspergillus species and opens up new opportunities in the field of diagnostics of fungal infections.

Understanding solid nitrogen through molecular dynamics simulations with a machine-learning potential

We construct a fast, transferable, general purpose, machine-learning interatomic potential suitable for large-scale simulations of N2. The potential is trained only on high quality quantum chemical molecule-molecule interactions; no condensed phase information is used. Although there are no explicit or implicit many-molecule interaction terms, the potential reproduces the experimental phase diagram including the melt curve and the molecular solid phases of nitrogen up to 10GPa. This demonstrates that many-molecule interactions are unnecessary to explain the condensed phases of N2. With increased pressure, transitions are observed from cubic (α), which optimizes quadrupole-quadrupole interactions, through tetragonal (γ), which allows more efficient packing, to monoclinic (λ), which packs still more efficiently. On heating, we obtain the hcp three-dimensional (3D) rotor phase (β) and, at pressure, the cubic δ phase which contains both 3D and 2D rotors, tetragonal δ* phase with 2D rotors, and the rhombohedral ε. Molecular dynamics demonstrates where these phases are indeed rotors, rather than frustrated order. The model supports the metastability of the complex ι phase, but not the reported existence of the wide range of bond lengths. The thermodynamic transitions involve both shifts of molecular centers and rotations of molecules: the onset of rotation is rapid, whereas motion of molecular centers is inhibited and we suggest that this is the cause of the experimentally observed sluggishness of transitions. Routine density functional theory calculations give a similar picture to the potential. Published by the American Physical Society 2024

Enhancing SARS-CoV-2 Lineage Surveillance through the Integration of a Simple and Direct qPCR-Based Protocol Adaptation with Established Machine Learning Algorithms.

Emerging and evolving Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) lineages, adapted to changing epidemiological conditions, present unprecedented challenges to global public health systems. Here, we introduce an adapted analytical approach that complements genomic sequencing, applying a cost-effective quantitative polymerase chain reaction (qPCR)-based assay. Viral RNA samples from SARS-CoV-2 positive cases detected by diagnostic laboratories or public health network units in Ceará, Brazil, were tracked for genomic surveillance and analyzed by using paired-end sequencing combined with integrative genomic analysis. Validation of a key structural variation was conducted with gel electrophoresis for the presence of a specific open reading frame 7a(ORF7a) gene deletion within the "BE.9" lineages tracked. The analytical innovation of our method is the optimization of a simple intercalating dye-based qPCR assay through repositioning primers from the ARTIC v4.1 amplicon panel to detect large molecular patterns. This assay distinguishes between "BE.9" and "non-BE.9" lineages, particularly BQ.1, without the need for expensive probes or sequencing. The protocol was validated against lineage predictions from next-generation sequencing (NGS) using 525 paired samples, achieving 93.3% sensitivity, 95.1% specificity, and 92.4% agreement, as measured by Cohen's Kappa coefficient. Machine learning (ML) models were trained using the melting curves from intercalating dye-based qPCR of 1724 samples, enabling highly accurate lineage assignment. Among them, the support vector machine (SVM) model had the best performance and after fine-tuning showed ∼96.52% (333/345) accuracy in comparison to the test data set. Our integrated approach provides an adapted analytical method that is both cost-effective and scalable, suitable for rapid assessment of emerging variants, especially in resource-limited settings. In this work, the protocol is applied to improve the monitoring of SARS-CoV-2 sublineages but can be extended to track any key molecular signature, including large insertions and deletions (indels) commonly observed in pathogenic agent subtypes. By offering a complement to traditional sequencing methods and utilizing easily trainable machine learning algorithms, our methodology contributes to enhanced molecular surveillance strategies and supports global efforts in pandemic control.

Pharmacogenetic assessment of cytostatic therapy efficacy depending on the polymorphism of T1 and M1 glutathione-S-transferase genes in breast cancer patients

Aim. To study the effect of deletion polymorphism of the GST genes (GSTT1, GSTM1) on chemotherapy (CT) efficacy in women diagnosed with breast cancer (BC) in the Primorsky Krai. Materials and methods. The study involved 132 women with breast cancer aged 23 to 79 years (mean age 48 ± 13 years) who received chemotherapy treatment. The detection of deletion (null) genotypes of GSTM1 and GSTT1 was carried out using multiplex PCR followed by an analysis of the melting curves of the reaction products. Results. Relapse-free survival (RFS) of BC patients with the “null” GSTT1 genotype was statistically significantly higher (116.7 months (9.6 years) versus 75.8 months (6.2 years)) than in patients with the “normal” GSTT genotype. Thus, in the case of the GSTT1“null” genotype, the risk of disease relapse decreased by 2.4 times (HR = 0.418, CI = 0.191–0.915, p = 0.024). Similar to GSTT1, the DFS of patients with the GSTM1 null genotype increased from 72.7 months (6 years) to 76.1 months (6.3 years). At the same time, the risk of disease relapse in carriers of the “null” GSTM1 genotype decreased by 1.7 times (HR = 0.596, CI = 0.369–0.964, p = 0.033) compared with that in carriers of the “normal” GSTM1 genotype. Conclusions. Deletion polymorphism of the GSTT1 and GSTM1 genes has a significant impact on chemotherapy efficacy in breast cancer patients. Carriers of “null” genotypes demonstrated a higher RFS and a lower risk of developing disease relapse. Further research in this direction and validation of the data obtained may contribute to individualizing the treatment of breast cancer patients, optimizing chemotherapy regimens, and reducing the number of adverse events.

Organophosphorus Flame Retardants and Metabolic Disruption: An in Silico, in Vitro, and in Vivo Study Focusing on Adiponectin Receptors.

Environmental chemical exposures have been associated with metabolic outcomes, and typically, their binding to nuclear hormone receptors is considered the molecular initiating event (MIE) for a number of outcomes. However, more studies are needed to understand the influence of such exposures on cell membrane-bound adiponectin receptors (AdipoRs), which are critical metabolic regulators. We aimed to clarify the potential interactions between AdipoRs and environmental chemicals, specifically organophosphorus flame retardants (OPFRs), and the resultant effects. Employing in silico simulation, cell thermal shift, and noncompetitive binding assays, we screened eight OPFRs for interactions with AdipoR1 and AdipoR2. We tested two key events underlying AdipoR modulation upon OPFR exposure in a liver cell model. The Toxicological Prioritization Index (ToxPi)scoring scheme was used to rank OPFRs according to their potential to disrupt AdipoR-associated metabolism. We further examined the inhibitory effect of OPFRs on AdipoR signaling activation in mouse models. Analyses identified pi-pi stacking and pi-sulfur interactions between the aryl-OPFRs 2-ethylhexyl diphenyl phosphate (EHDPP), triphenyl phosphate (TPhP), and tricresyl phosphate (TCP) and the transmembrane cavities of AdipoR1 and AdipoR2. Cell thermal shift assays showed a rightward shift in the AdipoR proteins' melting curves upon exposure to these three compounds. Although the binding sites differed from adiponectin, results suggest that aryl-OPFRs noncompetitively inhibited the binding of the endogenous peptide ligand ADP355 to the receptors. Analyses of key events underlying AdipoR modulation revealed that glucose uptake was notably lower, whereas lipid content was higher in cells exposed to aryl-OPFRs. EHDPP, TCP, and TPhP were ranked as the top three disruptors according to the ToxPi scores. A noncompetitive binding between these aryl-OPFRs and AdipoRs was also observed in wild-type (WT) mice. In db/db mice, the finding of lower blood glucose levels after ADP355 injection was diminished in the presence of a typical aryl-OPFR (TCP). WT mice exposed to TCP demonstrated lower AdipoR1 signaling, which was marked by lower phosphorylated AMP-activated protein kinase (pAMPK) and a higher expression of gluconeogenesis-related genes. Moreover, WT mice exposed to ADP355 demonstrated higher levels of pAMPK protein and peroxisome proliferator-activated receptor- messenger RNA. This was accompanied by higher glucose disposal and by lower levels of long-chain fatty acids and hepatic triglycerides; these metabolic improvements were negated upon TCP co-treatment. In silico, in vitro, and invivo assays suggest that aryl-OPFRs act as noncompetitive inhibitors of AdipoRs, preventing their activation by adiponectin, and thus function as antagonists to these receptors. Our study describes a novel MIE for chemical-induced metabolic disturbances and highlights a new pathway for environmental impact on metabolic health. https://doi.org/10.1289/EHP14634.

High-Resolution Melting Assay to Detect the Mutations That Cause the Y132F and G458S Substitutions at the ERG11 Gene Involved in Azole Resistance in Candida parapsilosis.

Candida parapsilosis is a pathogenic yeast that has reduced susceptibility to echinocandins and ranks as the second or third leading cause of candidaemia, depending on the geographical region. This yeast often causes nosocomial infections, which are frequently detected as outbreaks. In recent years, resistance to azoles in C. parapsilosis has increased globally, primarily due to the accumulation of mutations in the ERG11 gene. In this study, we have developed an assay based on real-time PCR and high-resolution melting (HRM) curve analysis to detect two of the most prevalent mutations at ERG11 that confer resistance to fluconazole (Y132F and G458S). We designed allele-specific oligonucleotides that selectively bind to either the wild type or mutated sequences and optimised the conditions to ensure amplification of the specific allele, followed by detection via high-resolution melting (HRM) analysis. The designed oligonucleotides to detect the Erg11Y132F and Erg11G458S mutations produced specific amplification of either WT or mutated alleles. We conducted a duplex real-time PCR combining oligonucleotides for the wild-type sequences in one mix, and oligonucleotides for the mutated alleles in another. Following this, we performed an analysis of the HRM curve to identify the amplified allele in each case. This technique was blindly evaluated on a set of 114 C. parapsilosis isolates, all of which were unequivocally identified using our approach. This technique offers a new method for the early detection of azole resistance mechanism in C. parapsilosis.

Rapid Detection of SLCO1B1 Polymorphisms Using Duplex Fluorescence Melting Curve Analysis: Implications for Personalized Drug Dosing in Clinical Settings.

The polymorphism of the solute carrier organic anion transporter family member 1B1 (SLCO1B1) gene exerts a marked influence on drug transport, thus playing a pivotal role in personalized drug dosing. This study endeavours to establish a rapid, precise, and straightforward method for detecting SLCO1B1 genetic variants utilizing Duplex Fluorescence Melting Curve Analysis (DFMCA). Whole blood samples were collected from 54 individuals from Meizhou People's Hospital (2023.01-2023.03), with a mean age of 58.90 years (SD = 7.86), including 28 men and 26 women. DNA was extracted from these samples and subjected to PCR amplification targeting two allelic regions. Primers, fluorescent probes, and corresponding allelic target sequences were designed specifically for two common SLCO1B1 polymorphisms (rs2306283 and rs4149056). The functionality of the fluorescent probes in binding to their respective allelic targets was verified using melting curve analysis, enabling the identification of distinct melting temperatures for different genotypes. Subsequently, DFMCA was employed to differentiate genotypes based on the melting temperature shifts of the corresponding fluorescent probes. The sensitivity, accuracy, and consistency of the method were evaluated, with sequencing validation performed on a subset of samples. DFMCA facilitated the concurrent detection and accurate genotyping of both polymorphisms within 2hours, demonstrating concordance with sequencing results from randomly selected samples. Importantly, stable detection performance was achieved for human genomic DNA at concentrations ≥ 3.125 ng. In a cohort comprising Han Chinese individuals from southern China, the allele frequencies for rs2306283 (A: 28.7%, G: 71.3%) and rs4149056 (T: 88.89%, C: 11.11%) concurred well with previous studies in the Han Chinese population. The SNP typing system utilizing DFMCA technology presents advantages in terms of speed, ease of use, accuracy, and cost-effectiveness, making it a suitable tool.

The pertinence of resistin gene single nucleotide polymorphism G > A and its expression in oral cancer

Background/aimOral cancer (OC) is the leading cause of fatalities in Pakistan among males due to inadequate oral hygiene and chewing habits. However, genetic susceptibility patterns also play a critical role in disease progression. Since the frequency of Resistin (RETN) SNP (Single nucleotide polymorphism) rs3219175 is unknown; there is a requirement for early diagnosis of the OC. Therefore, the current study aims to determine the frequency of targeted SNP and develop a safe, simple, and fast alternative technique for better treatment using a real-time PCR assay with HRM (high-resolution melting curve) analysis. Materials and methodsA case-control study was conducted on 35 Oral squamous cell carcinomas (OSCC) diagnosed patients and 35 healthy individuals. HRM and RT-PCR results were analysed by the bioinformatics analyses. ResultsThe frequency of RETN SNP rs3219175 genotypes GG and GA in male patients was 16 (46 %) and 5 (14 %) respectively and in females 8 (23 %) and 6 (17 %) respectively. The chi-square test of independence consummated the assessment between males and females in both control and patients. The relation between these variables was significant (p < 0.05). The interaction network of String 8.3 demonstrates strong interactions at a high confidence score, which helps to characterize functional disorders that may be a causative factor for oral pathology. Reactome and KEGG data were acquired to rule out the pathway involvement of the targeted gene. MuPIT software was used to identify the 3D structure or RETN and their expected mutation effect. ConclusionThis study provides baseline data regarding the frequency of RETN SNP rs3219175 among the Pakistani population. For further clarification of their stage in cancer emergence and growth, large-scale studies must be conducted. This study might be helpful in the precision medicine approach and provide better therapeutic for OSCC patients.

Effects of Dietary Protein Levels, Net Energy Levels, and Essential Amino Acid-to-True Protein Ratios on Broiler Performance.

Supplementing essential amino acids (EAAs) without considering non-EAA (NEAA) and energy contents in reduced-crude protein (CP) diets may alter EAA-to-true protein (E:T) and energy-to-protein ratios, potentially compromising growth. This study aimed to evaluate the effects of CP, E:T, and net energy (NE) on broiler performance. The treatments were as follows: T1-reduced CP (16%, RP), low NE (9.9 MJ/kg, LNE), low E:T (0.56, LE:T); T2-RP, LNE, high E:T (0.60, HE:T), with imbalanced EAA (excess Met and deficient Thr); T3-RP, high NE (10.4 MJ/kg, HNE), LE:T; T4-RP, HNE, HE:T; T5-normal CP (18%, NP), LNE, LE:T; T6-NP, LNE, HE:T; T7-NP, HNE, LE:T; and T8-NP, HNE, HE:T. The study employed as-hatched Cobb 500 broilers in two experiments. Exp.1 studied the broiler performance from d19 to 35, with eight replicates per treatment and 16 birds per replicate (n = 1024). Birds were randomly assigned to different treatments, and at the end of their lives, all were dissected to determine their sex and account for any gender-related effects. Exp.2 measured NE values in respiration chambers from d25 to 28, with six replicates per treatment, and two birds (a male and a female) per replicate (n = 96). The bird gender was determined through high-resolution melting curve analysis of feather DNA. The measured NE values were used to calculate NE intake (NEi) in Exp.1. The results showed that T4 improved (p < 0.001) weight gain (WG), feed conversion ratio (FCR), and NEi relative to T1, T2, and T3, and protein efficiency (WG/CP intake) relative to all treatments. The live performance (feed intake, WG, FCR) and NEi of birds fed T4 reached a level equal to those of birds fed NP diets (T5 to T8). These results suggest that a dietary E:T ratio of 0.60 is necessary to maximize nutrient utilization and to restore growth rate in broilers fed RP diets.

Design of a duplex Loop-mediated isothermal amplification assay suitable for real-time, and end-point, colorimetric detection of Vibrio parahaemolyticus, and a universal Internal Amplification Control, in mussel samples

Vibrio parahaemolyticus remains as one of the most important foodborne pathogens worldwide, causing gastrointestinal disease in humans due to the consumption of raw, or undercooked, seafood. Even though molecular techniques, like PCR and qPCR, have allowed to overcome most of the limitations associated to culture-based methods, some persist such as the need for highly trained personnel, or the need to acquire expensive equipment. Loop-mediated isothermal amplification, LAMP, has emerged as a promising tool to solve these issues. The present study reports the development of a novel LAMP-based method for the detection of V. parahaemolyticus in mussels. This assay targets the gene toxR, and implements a novel, universal, freely available Internal Amplification Control (IAC) which was detected in a multiplex real-time LAMP format, where it is differentiated by melt curve analysis (86.70 ± 0.32 °C and 90.24 ± 0.34 °C for toxR and IAC respectively) and did not impact the analytical sensitivity of the target. Alternatively, the IAC was also applied in a parallel reaction and the assays performed by end-point, colorimetric LAMP. This novel IAC allowed to identify reaction inhibition due to matrix effect, and rule out false negative results. This new assay was combined with the ISO 21872 enrichment protocol, and the evaluation of spiked mussel samples indicated that an LOD95 of 14.5 and 10.8 CFU/25g could be reached after the primary 6h and secondary 24h enrichment steps by real-time LAMP, and 27.9 and 10.9 CFU/25g by naked-eye, color LAMP. It was important to note that in addition to these differences in terms of LOD95, a higher number of samples presenting reaction inhibition was observed after the primary enrichment, compared to the secondary, all successfully identified thanks to the new IAC; and also that statistically longer amplification times were required (Cq values of 19.47 ± 7.87 and 7.90 ± 1.20 respectively). The overall evaluation of the method returned values of diagnostic sensitivity, specificity and accuracy of 100 % along with an index of concordance of 1.00. These results were consistent regardless if the real-time, or the colorimetric, LAMP strategy was followed.

Rapid identification, pathotyping and quantification of infectious bursal disease virus by high-resolution melting curve quantitative reverse transcription PCR analysis: An innovative technology well-suited for real-time large-scale epidemiological surveillance

With the virus continuing to evolve, very virulent IBDV (vvIBDV) and novel variant IBDV (nvIBDV) have become the predominant epidemic strains in China, exacerbated by the widespread use of attenuated vaccine strains (attIBDV), making a complex infection situation of IBDV in the field. Therefore, developing a rapid and accurate high-resolution melting curve quantitative reverse transcription PCR (HRM-qRT-PCR) for the identification and pathotyping of IBDV is crucial for clinical monitoring and disease control. Extensive data analysis and genome-screening of the three dominant IBDV pathotypes identified a specific region (nucleotides 2450–2603 in segment A) with distinct GC content as the detection target. Experimental testing of HRM-qRT-PCR revealed distinct melting curves and high sensitivity, with the detection limits of 61.2 copies/μL, 61.1 copies/μL and 67.5 copies/μL for vvIBDV, nvIBDV and attIBDV, respectively. The method exhibited excellent specificity, with no inter-genotypes cross-reactivity among the three pathotypes and no reactivity to other common avian pathogens. Applied to samples with double and triple co-infections of different IBDV pathotypes, the method displayed specific melting peaks corresponding to the viruses present in the samples, with an accuracy rate of 100 %. This method precisely identifies and differentiates all the single or co-infected samples, generating distinct peaks corresponding to the Tm values of each virus pathotype in traditional melting curve plots. Furthermore, the method overcomes the limitations of traditional pathotyping methods, requiring only one reaction to achieve rapid viral pathotyping and facilitating quantitative analysis of viruses within the samples. This study introduces an innovative HRM-qRT-PCR method, offering new technology to rapid and accurate identification, pathotyping and quantification of vvIBDV, nvIBDV, and attIBDV. With strong discriminatory power, user-friendliness and a short processing time, this method is highly attractive for the rapid IBDV pathotyping in real-time large-scale epidemiological surveillance during outbreaks.

Investigation on Melting Curves and Phase Diagrams for CaO3 Using Deep Learning Potentials.

Melting in the deep rocky parts of planets plays an important role in geological processes such as planet formation, seismicity, magnetic field generation, thermal convection, and crustal evolution. Such processes are the key way to understanding the dynamics of planetary interiors and the history as well as mechanisms of planetary evolution. We herein investigate the melting curves and pressure-temperature (P-T)-phase diagrams for CaO3, a candidate mineral for the lower mantle, by means of the deep learning potential model. Using first-principles, molecular dynamics, and quasi-harmonic approximation, the reliability of the deep learning potential model is verified by calculating the high-temperature and high-pressure equations of state and phase transition pressures for the orthorhombic and tetragonal structures of CaO3 described by space groups Aea2 and P4̅21m, respectively. The melting temperatures of 975, 850, and 755 K at zero pressure are obtained by the single-phase, void, and two-phase methods, respectively, and their melting temperatures are analyzed by the radial distribution function and mean-square displacement to analyze the melting process. Finally, the melting phase diagrams of CaO3 at 0-135 GPa were obtained by the two-phase method.