Melting Curve Analysis Research Articles

Detection and subtyping of Blastocystis sp. in human and animal stool samples using high-resolution melting analysis

ObjectiveThis study aimed to investigate the prevalence and subtype distribution of the genus Blastocystis using high-resolution melting curve analysis (HRM), a novel molecular technique. Blastocystis sp., a unicellular, anaerobic, and zoonotic parasite, is commonly found in the digestive tracts of humans and animals. Transmission occurs via the fecal-oral route, with prevalence rates ranging from 20% in developed countries to over 60% in developing regions. Although the pathogenicity of Blastocystis remains debated, it has been detected in both symptomatic and asymptomatic individuals. Traditionally, direct microscopy with wet mounts is used for the rapid identification of Blastocystis in stool samples. However, molecular diagnostics have become essential for the accurate detection and differentiation of Blastocystis subtypesMaterials and methodsWe collected 730 stool samples from humans and domestic animals. Initial screening for Blastocystis sp. was performed using direct microscopy. Negative samples were cultured in a two-phase culture medium and re-examined after 2–3 days. HRM analysis was then employed to identify and differentiate Blastocystis subtypes using specific primers via real-time PCR.ResultsSix subtypes were identified, with ST7 (30%) being the most prevalent, followed by ST3 (28%), ST2 (16%), ST1 (14%), ST5 (6%), and ST14 (6%). In humans, ST3 was the most common subtype, also found in poultry and sheep, whereas ST7 was mainly detected in domestic animals. Notably, ST1-ST3 were identified in domesticated animals, indicating a pattern of cross-species transmission.ConclusionST3 (28%) and ST7 (30%) were the most prevalent subtypes across all samples, with distinct distributions in human and animal hosts. The HRM technique demonstrated efficiency and cost-effectiveness, providing a rapid and accurate method for Blastocystis subtype identification in developing countries, which can expedite diagnostic responses and reduce the need for sequencing. This supports the potential for HRM to improve epidemiological surveillance and understanding of cross-species transmission.

Unexpected recombination at the polled locus in a horned Holstein calf from the mating of a homozygous polled sire and a heterozygous polled cow.

In this study, I report an unexpected case of a Holstein calf that developed horns even though the sire was homozygous and the dam was heterozygous for polledness. After verifying and confirming the correct parentage, the parents and offspring were genotyped with the Illumina EuroG_MD BeadChip and the SNPs in the polled region on chromosome 1 were evaluated. In addition, the father was sequenced with next generation sequencing to identify possible, previously unknown variants. The deletion of two base pairs within the causative 80-kb duplication described for the Friesian polled variant was verified by melting curve analysis and the 80-kb duplication by droplet digital PCR. Analysis of all data showed that, as expected, the calf was heterozygous for all SNP positions flanking the 80-kb duplication but was homozygous wild type in the 80-kb duplication region and therefore carried horns. This is certainly a very rare case of a recombination within the highly conserved polled region, which on the one hand confirms that only the 80-kb duplication is responsible for the expression of the Friesian polled variant, but on the other hand also shows that caution is required when interpreting the usual routine genotyping of the horn status based on linked single nucleotide polymorphisms in the polled region on chromosome 1. Based on the present case, it is recommended that, in addition to the evaluation of the SNP data of the BeadChip, an extended diagnosis with direct detection of the 2-bp deletion (1:g.2629156_2629158delGT) should be carried out in any case when detecting the Friesian polled variant.

Integration of a high-resolution melt curve assay into a commercial quantification kit for preliminary identification of biological mixtures.

In recent years, DNA analysis techniques have drastically increased in sensitivity, allowing for low template DNA samples to be more easily detected and used for identification. Since the problems inherent in low template DNA are exacerbated in DNA mixture samples, it would be advantageous to incorporate an assay earlier in the DNA workflow that could detect a mixture and, potentially, determine the number of contributors. Some real-time PCR instruments have high-resolution melt curve analysis (HRM) capabilities, allowing for an opportunity to integrate an HRM screening assay into a commercial DNA quantification kit. This work describes the integration of a mixture screening HRM assay using STR loci D5S818 and D18S51 into Qiagen's Investigator Quantiplex® kit. The integrated Quantiplex®-HRM assay was tested on two qPCR platforms: The Rotor-Gene® Q and the QuantStudio™ 6 Flex. Data from this assay was analyzed using linear discriminant and support vector machine analyses for sample classification. When HRM curve data from the Rotor-Gene® Q was used for classification, the integrated assay exhibited an overall accuracy of 89.39%, correctly classifying 87.5% of single source samples and 100% of mixtures. When HRM curve data from the QuantStudio™ 6 Flex was used for classification, the integrated assay exhibited an overall accuracy of 87.88%, correctly classifying 87.5% of single source samples and 90% of mixtures. The overall accuracy of the integrated Quantiplex®-HRM assay on both instruments met our goal of ≥ 80% accuracy, demonstrating the viability of the assay to detect mixtures when integrated into a commercial quantification kit.

Application of 2D polymerase chain reaction for single-tube detection of high-risk human papillomaviruses

PurposeThe persistent infection of high-risk HPV (HR-HPV) is intricately linked to the onset and progression of cervical cancer. This research endeavored to develop a high-throughput 2D PCR method for closed-tube genotyping of 11 HR-HPVs.MethodsBase-quenched probes were specifically designed for FAM, VIC, and CY5 channels. The 2D PCR system underwent optimization, with its detection performance assessed in terms of specificity and sensitivity. Plasmid mixtures was used to simulate multiple infections of HPV, providing preliminary insights into the detection efficacy and throughput of the 2D PCR technology. Ultimately, the detection capability of this method was assessed using clinical samples.ResultsThe sequenced tags, when paired with primers, could generate Tm differences exceeding 3°C. These were then integrated with a fluorescent channel and Tm to differentiate and identify target genes upon detection. The refined 2D PCR system was confirmed to be free from cross-reactions and exhibited high specificity, capable of detecting 12 target genes within a single tube. A total of 294 cervical exfoliated cell samples were tested using 2D PCR and flow fluorescence hybridization method. The overall concordance between the two detection methods was 96.17% (Kappa = 0.910).ConclusionThe 2D PCR method, which integrates asymmetric PCR amplification with melting curve analysis, has the capacity to detect 11 types of HR-HPVs across three channels. This closed-tube detection approach offers several benefits including high throughput, straightforward operation, and low detection cost. Consequently, it can be effectively utilized for early screening and prevention of cervical cancer.

The role of isoniazid dosage and NAT2 gene polymorphism in the treatment of tuberculous meningitis.

Tuberculous meningitis (TBM) is a non-purulent inflammatory condition affecting the meninges and spinal membranes, caused by Mycobacterium tuberculosis (MTB) infection. This study seeks to explore the impact of varying INH dosages and NAT2 gene polymorphisms on TBM treatment, contributing new insights to improve clinical management and patient prognosis. Patients with TBM hospitalized between July 2020 and December 2022 were categorized into two groups based on INH dosage: the standard-dose group (300 mg/day) and the high-dose group (600 mg/day). General and baseline data were collected, and NAT2 genotypes were identified using real-time fluorescent PCR with melting curve analysis. The clinical characteristics and outcomes of patients with TBM under varying INH dosages were analyzed. This study enrolled 119 patients with TBM, including 32 (26.9%) in the standard-dose group and 87 (73.1%) in the high-dose group. The NAT2 genotypes were distributed as follows: 34 (28.6%) fast acetylators (FA), 73 (61.3%) intermediate acetylators (IA), and 12 (10.1%) slow acetylators (SA). By month 12, 25 patients (21.0%) experienced disability or death, with 22 cases (18.5%) occurring by the end of the 3rd month. Disability and mortality rates differed significantly between the standard-dose and high-dose groups for IA-type TBM patients (P = 0.014). Univariate analysis showed significant differences between groups in baseline focal neurological impairment and disability or mortality by the 3rd and 12th months. Multivariate logistic regression identified INH dosage, cranial nerve palsy, age, and headache as key prognostic factors for TBM. High-dose INH treatment was associated with a reduced incidence of disability or death compared to the standard-dose regimen, indicating better efficacy and prognosis. In patients with IA-type TBM, the high-dose group showed a significantly lower rate of disability or mortality, suggesting that higher INH dosages may reduce the risk of adverse outcomes.

Digital Melting Curve Analysis for Multiplex Quantification of Nucleic Acids on Droplet Digital PCR.

We present a cost-effective and simple multiplex nucleic acid quantification method using droplet digital PCR (ddPCR) with digital melting curve analysis (MCA). This approach eliminates the need for complex fluorescent probe design, reducing both costs and dependence on fluorescence channels. We developed a convolutional neighborhood search algorithm to correct droplet displacement during heating, ensuring precise tracking and accurate extraction of melting curves. An experimental protocol for digital MCA on the ddPCR platform was established, enabling accurate quantification of six target pathogen genes using a single fluorescence channel, with an average accuracy of 85%. Our method overcomes the multiplexing limitations of ddPCR, facilitating its application in multi-target pathogen detection.

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

The purpose of the research was to examine the prevalence rates of screening for genetics and hearing simultaneously in neonates and provide scientific evidence for the beneficial application of newborn screening in the Southeast China population. Between June 2015 and March 2023, 27,843 newborns were enrolled in the study. All participants were screened by otoacoustic emissions at 2days of age. Fifteen variant hotspots in the four deafness genes (GJB2, GJB3, SLC26A4 and MTRNR1) were detected using multicolour melting curve analysis. Newborn screening data were also analysed. In otoacoustic emissions testing, 244 newborns (0.88%) failed the secondary screening. According to genetic testing, 1307 (4.69%) newborns had at least one variant. GJB2 c.235delC (2.34%) and SLC26A4 c.919-2A>G (0.91%) were the major deafness-related variants in the Wenzhou area of Southeast China. In addition, a difference in the number of newborns with variants was observed between the passed and failed groups. The difference in the positive rate between the two groups was statistically significant (χ2=274.969, P<0.05). Newborn screening for deafness genes by cord blood-based melting curve analysis can be applied to genetic counselling, prenatal diagnosis, and genetic screening of newborns with sensorineural hearing impairment with an unknown cause. The new PCR melting curve analysis approach is more effective and more convenient than SNaPshot and MS-based assay testing. Furthermore, it has a lower cost and is more suitable for clinical testing.

Melting curve analyses in the quantitative real-time polymerase chain reaction of methylated/non-methylated DNA toward the detection of oral cancer using gargle fluid

Melting curve analyses in the quantitative real-time polymerase chain reaction of methylated/non-methylated DNA toward the detection of oral cancer using gargle fluid

Evaluation of a Commercial Multiplex Real-Time PCR with Melting Curve Analysis for the Detection of Mycobacterium tuberculosis Complex and Five Nontuberculous Mycobacterial Species.

Accurate and timely diagnosis of mycobacterial infections, including Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM), is crucial for effective disease management. This study evaluated the performance of the NeoPlex TB/NTM-5 Detection Kit (NeoPlex assay, Seongnam, Republic of Korea), a multiplex real-time PCR assay that incorporates melting curve analysis, compared with the line-probe assay (LPA). The NeoPlex assay could simultaneously detect and differentiate MTBC from five other NTM species: Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium abscessus, and Mycobacterium massiliense. A total of 91 acid-fast bacillus culture-positive samples, comprising 36 MTBC and 55 NTM isolates, were collected from the Korea University Anam Hospital. The NeoPlex assay successfully detected nucleic acids in 87 of the 91 isolates (95.6%). Notably, it identified additional mycobacterial nucleic acids not detected by the LPA in eight isolates. These findings were confirmed via DNA sequencing. The assay had 100% sensitivity and specificity for M. intracellulare, M. abscessus, M. massilense, NTM, and MTBC, whereas it had 100% specificity and sensitivity of 90.9% and 75.0% for M. avium and M. kansasii, respectively. These results highlight the potential of the NeoPlex assay to enhance rapid and accurate diagnosis of mycobacterial infections, particularly in settings in which prompt treatment initiation is essential.

DNA Mimic Foldamer Recognition of a Chromosomal Protein

Helical aromatic oligoamide foldamers bearing anionic side chains that mimic the overall shape and charge surface distribution of DNA were synthesized. Their interactions with chromosomal protein Sac7d, a non‐sequence‐selective DNA‐binder that kinks DNA, were investigated by Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC), Circular Dichroism spectroscopy (CD), melting curve analysis, Atomic Force Microscopy (AFM), and Nuclear Magnetic Resonance (NMR), as well as by single crystal X‐ray crystallography. The foldamers were shown to bind to Sac7d better than a DNA duplex of comparable length. The interaction is diastereoselective and takes place at the DNA binding site. Crystallography revealed that the DNA mimic foldamers have a binding mode of their own and that they can bind to Sac7d without being kinked.

DNA Mimic Foldamer Recognition of a Chromosomal Protein.

Helical aromatic oligoamide foldamers bearing anionic side chains that mimic the overall shape and charge surface distribution of DNA were synthesized. Their interactions with chromosomal protein Sac7d, a non-sequence-selective DNA-binder that kinks DNA, were investigated by Surface Plasmon Resonance (SPR), Isothermal Titration Calorimetry (ITC), Circular Dichroism spectroscopy (CD), melting curve analysis, Atomic Force Microscopy (AFM), and Nuclear Magnetic Resonance (NMR), as well as by single crystal X-ray crystallography. The foldamers were shown to bind to Sac7d better than a DNA duplex of comparable length. The interaction is diastereoselective and takes place at the DNA binding site. Crystallography revealed that the DNA mimic foldamers have a binding mode of their own and that they can bind to Sac7d without being kinked.

Loop-Mediated Isothermal Amplification-Based Workflow for the Detection and Serotyping of Salmonella spp. in Environmental Poultry Flock Samples.

Salmonella spp. is one of the most important foodborne pathogens worldwide. Given the fact that poultry and poultry products are the main source of human infection, Salmonella control in these farms is of utmost importance. To better control this pathogen in farms, boot swabs are used to sample farm environments but the analysis of these swabs is mainly based on culture-dependent methods. In the present study, a novel loop-mediated isothermal amplification (LAMP) method was developed for the rapid screening of Salmonella spp. in boot swab samples from broiler flock environments. Four different DNA extraction protocols were evaluated in depth, including a simple thermal lysis, a chelex-based protocol and two thermal lysis protocols followed by the purification of magnetic beads made of silica ("glass milk") in order to determine the most suitable alternative for potential on-site, farm analyses. The methodology evaluation included a blind interlaboratory assay and as a proof-of-concept, a naked-eye colorimetric assay was also included. Following the final methodology, it was possible to reach an LoD50 of 1.8 CFU/25 g of the samples, with a high relative sensitivity (95.7%), specificity (100%) and accuracy (96.6%) along with Cohen's kappa of concordance with respect to the ISO standard 6579-1:2017 of 0.9, with an RLOD of 1.3. In addition to this, due to the relevance of certain serotypes with the genus Salmonella spp., a serotype LAMP panel for the specific identification of S. Typhimurium, S. Enteritidis, S. Infantis, S. Hadar and S. Virchow was also developed. Even though some degree of cross-reactivity among the primers developed was observed, all the serotypes could be accurately identified based on their melt curve analysis profile. Taken together, in the present study, a rapid Salmonella spp. screening method, suitable for farm applications, was developed, along with a serotyping panel that could be used in a laboratory setup for the identification of the most relevant serotypes of the genus, taking advantage of real-time amplification followed by melt curve analysis.

Differential Methylation of CYP11B1 in Girls with High DHEAS Levels and Correlation with 11-Oxyandrogen Levels: A Pilot Study

Introduction: Premature adrenarche in girls is defined biochemically by an increase in adrenal androgen (DHEAS) levels above the age-specific reference range before age 8 years. Recently, increased levels of 11-oxyandrogens have also been reported in girls with premature adrenarche. Epigenetic modifications, specifically CpG methylation, may affect gene expression and/or activity of steroidogenic enzymes during developmental changes in adrenal androgen secretion. Objective: The aim of the study was to determine whether circulating 11-oxyandrogen levels in post-menarcheal girls are associated with the methylation status of genes involved in 11-oxyandrogen steroidogenesis. Methods: Ninety-seven healthy girls followed since the age of 3 years were classified, according to DHEAS serum concentration at age 6–7 years, as normal DHEAS (<42 μg/dL [75th percentile for population]) or high DHEAS (≥42 μg/dL). At Tanner stage 2, the methylation status of CpG sites located in CYP11B1 and HSD11B2 genes was analyzed in genomic DNA from peripheral blood leukocytes by the melting curve analysis methylation assay. Eleven-oxyandrogen concentrations were assessed at 4 years post menarche. Results: Significantly lower methylation levels were detected in the CYP11B1 gene in girls with high versus normal serum DHEAS levels, with no differences found in HSD11B2 gene. Additionally, CYP11B1 methylation status correlated inversely with 11β-hydroxy-androstenedione and 11-ketotestosterone levels. Furthermore, CYP11B1 methylation in the full cohort correlated inversely with insulin concentration at Tanner 1 and with body mass index at Tanner stage 1 and 2. Conclusion: This pilot study proposes the hypothesis that a lower methylation of CYP11B1 may be a mechanism contributing to increased concentrations of 11-oxyandrogens in premature adrenarche and its associated metabolic risk.

Does Antibiotic Use Contribute to Biofilm Resistance in Sink Drains? A Case Study from Four German Hospital Wards.

Backgound. As biofilms are known to harbour (multi-)resistant species, their presence in health settings must be considered critical. Although there is evidence that bacteria spread from drains to the outside, there is still a lack of research data focusing on drain biofilms from hospitals. Methods. We sampled biofilms from various wards of Helios Hospital Krefeld (Germany), where comprehensive antibiotic consumption data were available. Biofilms were analysed by cell counting, isolation of relevant bacterial groups and genetic and phenotypical resistance parameters. Data were correlated with the prescribed antibiotics of the respective ward. Furthermore, an ex situ biofilm model was employed to investigate the influence of sub-inhibitory antibiotics on the bacterial community and the prevalence of class 1 integrons. Results. Our results show that every ward harboured medically relevant bacterial species. While no significant differences were found in cell counts, the median prevalence of the resistance marker gene intI1 correlated with the amount of prescribed antibiotics. In contrast, phenotypical resistances showed no similar tendency. In addition, melting curve analysis data and changes in intI1 prevalence show that the composition of the bacterial community shifted depending on the biofilm and antibiotic. Conclusions. To the best of our knowledge, our study is the first considering possible correlations between the consumption data of hospital wards and resistances in drain biofilms the way we did. Based on our results, we conclude that sub-inhibitory concentrations of antibiotics have no general effect on biofilms in terms of bacterial community shift and occurrence of antibiotic-resistant species. Amongst other things, the effect depends on the initial composition of the bacterial community, the antibiotic used and the intrinsic bacterial resistance, e.g., prevalence of class 1 integrons.

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.

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.

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.

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.

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.