Detection Of Polymorphisms Research Articles

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.

Optimization and Clinical Application Potential of Single Nucleotide Polymorphism Detection Method Based on CRISPR/Cas12a and Recombinase Polymerase Amplification.

Conventional methods for detecting single nucleotide polymorphisms (SNPs) in clinical practice often require substantial time, labor, and specialized equipment, limiting their widespread application. To address this limitation, we refined our previous SNP detection method, IMAS-RPA [introducing an extra mismatched base adjacent to the single-base mutant site by recombinase polymerase amplification (RPA)], resulting in an updated version termed IMAS-RPAv2. We began by introducing a suboptimal protospacer adjacent motif (PAM) sequence, GTTG, into the double-stranded DNA (dsDNA) products using either RPA or reverse transcription RPA. This modification decreased the efficiency with which CRISPR RNA (crRNA) recognizes the PAM and locally unwinds the dsDNA to form an R loop. After a delay, the R loop forms. However, due to the intentional incorporation of a mismatched base on the crRNA relative to the wild-type double-stranded DNA (WT-dsDNA), a continuous two-base mismatch is established between the crRNA and WT-dsDNA. Consequently, WT-dsDNA does not activate CRISPR/Cas12a's cleavage activity within a short time, while variant-type dsDNA continues to activate CRISPR/Cas12a and produce a robust fluorescence signal. This improvement significantly enhances the SNP discrimination sensitivity, allowing for detection at the single-copy level. Results were observed using both a conventional microplate reader and a specially designed portable device created through 3D printing. This device allows a direct fluorescence observation without the need for additional equipment. Consequently, the entire detection process becomes independent of large-scale equipment. This greatly expands its range of applications and offers promising prospects for clinical use.

Association of Celiac Disease and H. pylori Infection with ATG5 Polymorphism and Interleukin-33

Background: Celiac disease (CD) is an inflammatory small intestine autoimmune disease. The study aims to investigate the association and the detection of ATG5 polymorphism between celiac disease (CD) and H. pylori infection, and the association with Interleukin-33 (IL-33).Methods: The study groups included patients from Iraq, ages 4 to 35. Two primary patient groups were created: sixteen had positive H. pylori and celiac disease, and thirty had positive CD and negative H. pylori disease. The levels of tissue transglutaminase IgA (TTG IgA), H. pylori IgG, and IL-33 were measured using the ELISA method. The primers were amplified using PCR.Results: With celiac disease, the patient group's TTG IgA levels increased dramatically. The test also showed significant variations (P=0.054) in the H. pylori IgG levels between the patient and control groups. The H. pylori seropositivity test showed a statistically significant difference (p ≤0.033) between seropositive and seronegative individuals, while the patient group's IL-33 levels did not significantly differ (P ≤0.299) from the control group.Conclusions: Our results showed that CD is more common in women and occurs in the age range of 24-35 years. It also showed that the mutant variant of ATG5 is associated with CD, and the significance of H. pylori IgG serum levels in the patient group may indicate that the bacteria involved in CD. Furthermore, H. pylori infection is more strongly linked to serum IL-33 levels than CD.Keywords: Celiac disease; H. pylori; PCR; IL-33; ATG5 polymorphism

Personalized medicine beyond cancer: Impact on other diseases

Personalized medicine beyond cancer: Impact on other diseases With a focus on type 2 diabetes and Parkinson’s disease, Dr Priya Hays explores how personalized medicine approaches are impacting the development of therapies for other chronic conditions beyond cancer. Genetic testing and cancer outcomes have been considerably advanced by precision medicine, or personalized medicine, with gene panel testing conducted routinely in patient care and targeted therapy approaches incorporated into oncology. It may be argued that with these significant advances, diseases such as type 2 diabetes, cardiovascular disease, neurological diseases such as Parkinson’s disease, and Alzheimer’s disease and psychiatric diseases have been unaffected by the gains of personalized medicine. However, this is not entirely accurate as aspects of personalized medicine such as pharmacogenomics, targeted approaches for neurological systems, and single nucleotide polymorphism detection have resulted in safe and effective diagnostics and treatments for cardiovascular disease, diabetes and neurological disorders, and depression and schizophrenia. Outlined here are precision medicine approaches for type 2 diabetes and Parkinson’s disease.

Polymorphism detection and characterization of sperm cells chromatin remodeling associated genes in Murrah buffalo.

Seasonal variations significantly impact buffalo bull semen production and quality, particularly during the summer months. Understanding the genetic basis of these changes is important for managing bull fertility and improving sperm quality. The present study focused on characterizing and identifying polymorphisms in chromatin remodeling genes, protamines (PRMs) and Transition Nuclear Proteins (TNPs) in Murrah buffalo bulls with varying semen quality due to seasonal effects. Our findings revealed none of the coding region variation in PRM1, PRM2, TNP1, and TNP2, these genes are highly conserved in buffalo. Two intronic variants were identified, including G16C in PRM1 intron 1 and intronic SNP in PRM2 intron 1(G96A). The complete CDS of consensus sequence of bubaline PRM1 was 86.3% identical and 94.1% similar to the bovine PRM1. Whereas the complete CDS of consensus sequence of bubaline TNP2 was 78.2% identical and 91.0% similar to bovine TNP2. Further, no statistically significant differences in the fold change of TNP1, TNP2, PRM1, and PRM2 levels between the hot summer SNA and SA groups and the winter SNA and SA groups This study represents the first comprehensive report on the characterization of bubaline PRM1 (complete CDS), PRM2 (partial CDS), TNP1 (partial CDS), and TNP2 (complete CDS) genes in buffalo sperm cells. Results of the study, clearly indicate that the genes associated with protamine (PRM1 and TNP2) are highly conserved in Bubalus bubalis. Understanding these genetic underpinnings can have implications for improving buffalo bull fertility and semen quality.

Entropy-Driven Catalytic G-Quadruple Cycle Amplification Integrated with Ligases for Label-Free Detection of Single Nucleotide Polymorphisms.

G-Quadruplex/thioflavin (G4/THT) has become a very promising label-free fluorescent luminescent element for nucleic acid detection due to its good programmability and compatibility. However, the weak fluorescence efficiency of single-molecule G4/THT limits its potential applications. Here, we developed an entropy-driven catalytic (EDC) G4 (EDC-G4) cycle amplification technology as a universal label-free signal amplification and output system by properly programming classical EDC and G4 backbone sequences, preintegrated ligase chain reaction (LCR) for label-free sensitive detection of single nucleotide polymorphisms (SNPs). First, the positive strand LCR enabled specific transduction and preliminary signal amplification from single-base mutation information to single-strand information. Subsequently, the EDC-G4 cycle amplification reaction was activated, accompanied by the production of a large number of G4/THT luminophores to output fluorescent signals. The EDC-G4 system was proposed to address the weak fluorescence of G4/THT and obtain a label-free fluorescence signal amplification. The dual-signal amplification effect enabled the LCR-EDC-G4 detection system to accurately detect mutant target (MT) at concentrations as low as 22.39 fM and specifically identify 0.01% MT in a mixed detection pool. Moreover, the LCR-EDC-G4 system was further demonstrated for its potential application in real biological samples. Therefore, this study not only contributes ideas for the development of label-free fluorescent biosensing strategies but also provides a high-performance and practical SNP detection tool in parallel.

Assessing the therapeutic efficacy of artemether-lumefantrine for uncomplicated malaria in Lagos, Nigeria: a comprehensive study on treatment response and resistance markers

BackgroundThe burden of malaria persists in sub-Saharan Africa and the emergence of artemisinin resistance has introduced complexity to control efforts. Monitoring the efficacy of artemisinin-based treatment for malaria is crucial to address this challenge. This study assessed treatment efficacy of artemether-lumefantrine (AL) and genetic diversity of Plasmodium falciparum isolates in a Nigerian population.MethodsParticipants presenting with clinical symptoms of uncomplicated malaria at a health centre in Lagos, Nigeria, were screened for P. falciparum. Enrolled participants were treated with AL and monitored through scheduled check-up visits, clinical and laboratory examinations for 28 days. Parasite clearance and genetic diversity were assessed through polymerase chain reaction (PCR) analysis of merozoite surface proteins (msp1 and msp2). The prevalence of drug resistance mutations was assessed by P. falciparum multidrug resistance gene 1 (mdr1) genotyping followed by P. falciparum ubiquitin-specific protease 1 (ubp1) gene sequencing.ResultsThe PCR-uncorrected treatment outcome revealed 94.4% adequate clinical and parasitological response (ACPR) and 5.6% late parasitological failure (LPF) rates. After PCR correction, no suspected LPF case was detected and ACPR 67/67 (100%) was achieved in all the individuals. Moreover, a high prevalence of wild-type alleles for mdr1 N86Y (93.7%), and mdr1 D1246Y (87.5%) was observed. Genetic diversity analysis revealed predominant K1 allelic family for msp1 (90.2%) and FC27 for msp2 (64.4%). Estimated multiplicity of infection (MOI) was 1.7, with the highest MOI observed in the 5–15 years age group. ubp1 sequence analysis identified one nonsynonymous E1528D polymorphism at a low frequency (1.6%).ConclusionThe study demonstrated sustained efficacy of AL for treating uncomplicated P. falciparum malaria. Genetic diversity analysis revealed various allelic types, suggesting occurrences of polyclonal infections. Nonetheless, the detection of a significant ubp1 polymorphism could have future implications for the epidemiology of anti-malarial drug resistance in the population.

Systematic identification of minor histocompatibility antigens predicts outcomes of allogeneic hematopoietic cell transplantation.

T cell alloreactivity against minor histocompatibility antigens (mHAgs)-polymorphic peptides resulting from donor-recipient (D-R) disparity at sites of genetic polymorphisms-is at the core of the therapeutic effect of allogeneic hematopoietic cell transplantation (allo-HCT). Despite the crucial role of mHAgs in graft-versus-leukemia (GvL) and graft-versus-host disease (GvHD) reactions, it remains challenging to consistently link patient-specific mHAg repertoires to clinical outcomes. Here we devise an analytic framework to systematically identify mHAgs, including their detection on HLA class I ligandomes and functional verification of their immunogenicity. The method relies on the integration of polymorphism detection by whole-exome sequencing of germline DNA from D-R pairs with organ-specific transcriptional- and proteome-level expression. Application of this pipeline to 220 HLA-matched allo-HCT D-R pairs demonstrated that total and organ-specific mHAg load could independently predict the occurrence of acute GvHD and chronic pulmonary GvHD, respectively, and defined promising GvL targets, confirmed in a validation cohort of 58 D-R pairs, for the prevention or treatment of post-transplant disease recurrence.

G-quadruplex embedded in semi-CHA reaction combined with invasive reaction for label-free detection of single nucleotide polymorphisms

G-quadruplex/thioflavin T (G4/THT) is one of the ideal label-free fluorescent light-emitting elements in the field of biosensors due to its good programmability and adaptability. However, the unsatisfactory luminous efficiency of single-molecule G4/THT limits its more practical applications. Here, we developed a G4 embedded semi-catalytic hairpin assembly (G4-SCHA) reaction by rationally modifying the traditional CHA reaction, and combined with the invasive reaction, supplemented by magnetic separation technology, for label-free sensitive detection of single nucleotide polymorphisms (SNPs). The invasive reaction enabled specific recognition of single-base mutations in DNA sequences as well as preliminary signal cycle amplification. Then, magnetic separation was used to shield the false positive signals. Finally, the G4-SCHA was created for secondary amplification and label-free output of the signal. This dual-signal amplified label-free biosensor has been shown to detect mutant targets as low as 78.54 fM. What's more, this biosensor could distinguish 0.01 % of the mutant targets from a mixed sample containing a large number of wild-type targets. In addition, the detection of real and complex biological samples also verified the practical application value of this biosensor in the field of molecular design breeding. Therefore, this study improves a label-free fluorescent light-emitting element, and then proposes a simple, efficient and universal label-free SNP biosensing strategy, which also provides an important reference for the development of other G4/THT based biosensors.

Brain Glucose Metabolism and COMT Val 158 Met Polymorphism in Female Patients with Work-Related Stress.

Stress is a ubiquitous challenge in modern societies. Symptoms range from mood swings and cognitive impairment to autonomic symptoms. This study explores the link between work-related stress and the neurobiological element of brain processing, testing the hypothesis that patients with occupational stress have altered cerebral glucose consumption compared to healthy controls. The participants' present conditions were evaluated using an adapted WHO SCAN interview. Neural activity at rest was assessed by positron emission tomography (PET) with the glucose analogue [18F]fluorodeoxyglucose. Participants were genotyped for the Val158Met polymorphism of the COMT gene, believed to influence stress resilience. This study included 11 women with work-related stress and 11 demographically comparable healthy controls aged 28-62 years, with an average of 46.2 years. The PET scans indicated clusters of decreased glucose consumption primarily located in the white matter of frontal lobe sub-gyral areas in stress patients. COMT Val158Met polymorphism detection indicated no immediate relation of the homozygous alleles and stress resilience; however, healthy controls mainly had the heterozygous allele. In conclusion, the results support that work-related stress does affect the brain in the form of altered glucose metabolism, suggesting neurobiological effects could be related to white matter abnormalities rather than gray matter deterioration. Genotyping indicates a more complex picture than just that of the one type being more resilient to stress. Further studies recruiting a larger number of participants are needed to confirm our preliminary findings.

Genetic diversity analysis of Saccharosydne procerus in Hunan region, China.

Saccharosydne procerus serves as a significant alternative host for parasitoids of the important rice pest, rice planthoppers. Rearing S. procerus on the water bamboo plants near rice field can provide a parasitic site for parasitic wasps during the idle period of rice fields, thereby stabilizing the number of parasitoids and suppressing the number of rice planthoppers in the field. However, limited understanding of genetic diversity of S. procerus restricts its application. Therefore, this study aims to analyze the genetic diversity of S. procerus in Hunan region. In this study, 16 geographical populations of the S. procerus from the Hunan region were used. After screening, ISSR primers were employed for polymorphism detection. POPGENE32 software was used for genetic diversity analysis, and UPGMA clustering was applied for statistical analysis of different geographical populations to generate an evolutionary tree. Eleven ISSR primers were screened, resulting in the detection of 194 amplification locus, of which 126 were polymorphic. The average percentage of polymorphic locus was 64.95%. The mean Nei's gene diversity (H) was 0.2475, the mean Shannon's Information index (I) was 0.3708, and the Genetic diversity index among populations (Gst) was 0.3800. Cluster analysis identified three groups, with most populations concentrated in the second group, indicating no clear genetic structure. This suggests that the 16 populations of S. procerus exhibit high levels of genetic diversity.

A multi-AS-PCR-coupled CRISPR/Cas12a assay for the detection of ten single-base mutations

Single-nucleotide polymorphism (SNP) detection is critical for diagnosing diseases, and the development of rapid and accurate diagnostic tools is essential for treatment and prevention. Allele-specific polymerase chain reaction (AS-PCR) is widely used for detecting SNPs with multiplexing capabilities, while CRISPR-based technologies provide high sensitivity and specificity in targeting mutation sites through specific guide RNAs (gRNAs).In this study, we have integrated the high sensitivity and specificity of CRISPR technology with the multiplexing capabilities of AS-PCR, achieving the simultaneous detection of ten single-base mutations. As for Multi-AS-PCR, our research identified that competitive inhibition of primers targeting the same loci, coupled with divergent amplification efficiencies of these primers, could result in diminished amplification efficiency. Consequently, we adjusted and optimized primer combinations and ratios to enhance the amplification efficacy of Multi-AS-PCR. Finally, we successfully developed a novel nested Multi-AS-PCR-Cas12a method for multiplex SNPs detection. To evaluate the clinical utility of this method in a real-world setting, we applied it to diagnose rifampicin-resistant tuberculosis (TB). The limit of detection (LoD) for the nested Multi-AS-PCR-Cas12a was 102 aM, achieving sensitivity, specificity, positive predictive value, and negative predictive value of 100 %, 93.33 %, 90.00 %, and 100 %, respectively, compared to sequencing.In summary, by employing an innovative design that incorporates a universal reverse primer alongside ten distinct forward allele-specific primers, the nested Multi-AS-PCR-Cas12a technique facilitates the parallel detection of ten rpoB gene SNPs. This method also holds broad potential for the detection of drug-resistant gene mutations in infectious diseases and tumors, as well as for the screening of specific genetic disorders.

A bulged-type enzyme-free recognition strategy designed for single nucleotide polymorphisms integrating with label-free electrochemical biosensor

Compared to conventional nucleic acid detection methods, label-free single nucleotide polymorphism (SNP) detection presents challenging due to the necessity of discerning single base mismatches, especially in the field of enzyme-free detection. In this study, we introduce a novel bulged-type DNA duplex probe designed to significantly amplify single-base differences. This probe is integrated with programmable DNA-based nanostructures to develop a sensitive, label-free biosensor for nonenzymatic SNP detection. The duplex probe with one bulge could selectively identify wild-typed DNA (WT) and mutant-type DNA (MT) based on a competitive strand displacement reaction mechanism. The hyperbranched HCR (HHCR) by incorporating of hairpin DNA into the DNA tetrahedron and surface-tethering on the portable screen printing electrode (SPCE) significantly favor the formation of negatively charged DNA nanostructure. We harnessed strong repulsion of DNA nanostructure towards the electroactive [Fe(CN)₆]³⁻/⁴⁻ in combination with electrochemical technique to create a label-free biosensor. This simple, enzyme-free and label-free biosensor could detect MT with a detection limit of 56 aM, even in multiple sequence backgrounds. The study served as the proof-of-concept for the integration of enzyme-free competitive mechanism and label-free strategy, which can be extended as a powerful tool to various fields.

Rapid identification of Africanized honey bee Apis mellifera (hymenoptera: apidae) using high-resolution melting analysis DNA from honey

Genetic characterization of honey bee populations, particularly the identification of Africanized honey bees, has traditionally been based on different molecular methods that involve the amplification and analysis of different regions of the mitochondrial or nuclear genome which often take a long time. We introduce high-resolution melting analysis (HRM) as a novel approach for rapid and cost-effective identification of the African lineage in Apis mellifera populations from mitochondrial DNA extracted from honey. Samples were collected from 111 apiaries in 13 Argentinean provinces between 2012 and 2018. Total DNA was extracted from 10 g honey sediment and subjected to quantitative PCR (qPCR) using specific primers (APIS-F/Afr207R) amplifying a 207 bp product. Positive controls (Africanized/Non-Africanized) were used for mitotype validation and a subset of qPCR products were sequenced to compare different haplotypes. HRM analysis enabled the quick detection of sequence polymorphisms in short amplicons generated by qPCR. Among the analyzed apiaries, 26 located above 35° S in Misiones, La Rioja, Catamarca, Formosa, Corrientes, Córdoba, and Santa Fé exhibited the Africanized mitotypes while the remaining apiaries below this latitude, mainly in Buenos Aires, Rio Negro, Neuquén, and Entre Ríos, showed the European mitotypes. These results align with previous characterizations in the region. HRM analysis demonstrated high sensitivity and effectiveness, requiring minimal DNA input, and reducing costs and sampling efforts. This approach provides a rapid and reliable method for identifying the African lineage, facilitating honey bee breeding programs, conservation efforts, and management strategies in regions with Africanized honey bee populations.

Detection of single-nucleotide polymorphisms in growth hormone and insulin-like growth factor-1 genes related to growth traits in purebred and crossbred quails.

There is a limited amount of research conducted on quail breeding domestically and internationally, particularly at the molecular level. This study aimed to detect single-nucleotide polymorphisms in the growth hormone (GH) and insulin-like growth factor-1 (IGF-1) genes across two quail varieties and their hybrids correlate these genetic factors with body weight (BW) and growth rate at 0 and 6 weeks, and assess crossing effects. White and Japanese quail were crossed. Simultaneously producing pure varieties and crosses (genotypes) was achieved through this breeding strategy. Fifty females from each genotype were randomly selected for blood sampling. Genomic DNA was extracted and amplified from the blood using the DNeasy blood kit (Qiagen, Germany). Nucleotide polymorphism between quail genotypes was determined through DNA sequencing. Two types of alleles (A and B) for the GH gene in quails showed significant genotypic differences (AA, BB, and AB). The quail carried a mutated IGF-1 gene. For growth traits, substantial positive heterosis was detected. The genotype AA had the highest BW and weight gain. The white variety can act as a sire, and both white and Japanese varieties can function as dams to improve growth traits. The growth characteristics of the hybrids surpassed those of the original varieties.

Detection of Polymorphisms in FASN, DGAT1, and PPARGC1A Genes and Their Association with Milk Yield and Composition Traits in River Buffalo of Bangladesh.

This study aimed to identify SNPs in the intron, exon, and UTR regions of the FASN, DGAT1, and PPARGC1A genes and to investigate their possible association with milk yield and composition traits in the riverine buffalo of Bangladesh. A total of 150 DNA samples from riverine buffalo were used for PCR amplification with five pairs of primers, followed by association studies using a generalized linear model in R. SNP genotyping was performed by direct sequencing of the respective amplicon. Traits analyzed included DMY, fat%, protein%, and SNF%. This study identified 8 SNPs in FASN (g.7163G>A and g.7271C>T), DGAT1 (g.7809C>T and g.8525C>T) and PPARGC1A (g.387642C>T, g.387758A>G, g.409354A>G, and g.409452G>A). Genotypic and allelic frequencies differed significantly for each SNP genotype and did not follow the Hardy-Weinberg principle (p < 0.01 or p < 0.001) in most cases. The g.7163G>A and g.7271C>T SNP genotypes of the FASN gene were significantly associated with milk fat%, with the latter also significantly associated with SNF%. The g.8525C>T polymorphism of the DGAT1 gene significantly affected protein% (p < 0.01). Additionally, PPARGC1A gene polymorphisms showed significant associations: g.387642C>T with fat% (p < 0.05); g.387758A>G and g.409354A>G with protein% (p < 0.001) and SNF% (p < 0.01); and g.409452G>A with DMY (p < 0.001), fat% (p < 0.05), and protein% (p < 0.01). Reconstructed haplotypes of the PPARGC1A gene were significantly associated (p < 0.01) with all traits except SNF%. These findings suggest that polymorphisms in these three candidate genes have the potential as molecular markers for improving milk yield and composition traits in the riverine buffalo of Bangladesh.

Molecular study of Klebsiella pneumoniae antibiotic resistance genes isolates from wound infections

K. pneumoniae, which produces Carbapenemases, is of public health importance because of its resistance to antimicrobials. K. Pneumoniae is identified phenotypically by its large pink mucus appearance on MacConkey agar because of lactose fermentation and by bacteriological, microscopic, and biochemical tests using the VITK 2 device. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion. Klebsiella pneumoniae represented 5.3% (n = 16). It is highly resistant to antibiotics used to treat wound infections and shows the highest rate of resistance with cefepime 11(68.8%), followed by Ertapenem 9(56.3%), Doripenem and Imipenem 7(43.8%), and Meropenem 6(37.5%). Carbapenemases resistance was identified by phenotype through the Hodge test and also detection of Carbapenemases genes by polymerase chain reaction. The results showed the presence of the bla SHV 16 gene (100%) in K. pneumoniae isolates. The results showed that there were 4 isolates (25%) positive for the bla AMPC gene. The results also showed the presence of the bla KPC gene in two isolates (12.5%). The bla GES gene was not found in K. pneumoniae isolates. In general, the isolates were resistant to carbapenem and cefepime antibiotics. Five random samples were examined by publishing the results of the sequence analysis by BLAST in (NCBI) and the Mega 7 polymorphism detection program: it was found that there was no difference between the sequences, but rather the match was with the bacterium Klebsiella pneumoniae.

Development of a 45K pepper GBTS liquid-phase gene chip and its application in genome-wide association studies.

Pepper (Capsicum spp.) is a vegetable that is cultivated globally and has undergone extensive domestication, leading to a significant diversification in its agronomic traits. With the advancement of genomics in pepper and the reduction in sequencing costs, the high-throughput detection of single nucleotide polymorphisms (SNPs) and small insertions-deletions (indels) has become increasingly critical for analyzing pepper germplasms and improving breeding programs. As a result, there is a pressing need for a cost-effective, high-throughput, and versatile technique suitable for both foreground and background selection in pepper breeding. In the present study, Python-based web scraping scripts were utilized to systematically extract data from published literatures and relevant sequence databases focusing on pepper genomes. Subsequent to data extraction, SNPs and indels were meticulously identified and filtered. This process culminated in the delineation of core polymorphic sites, which were instrumental in the development of specific probes. Following this, comprehensive phenotypic and genotypic analyses were conducted on a diverse collection of 420 pepper germplasms. Concurrently, a genome-wide association study (GWAS) was conducted to elucidate the genetic determinants of helical fruit shape in peppers. In this study, a 45K pepper Genotyping-By-Target-Sequencing (GBTS) liquid-phase gene chip was developed on the GenoBaits platform. This chip is composed of 45,389 probes, of which 42,535 are derived from core polymorphic sites (CPS) in the background genetic landscape, while 2,854 are associated with foreground agronomic traits, spanning across 43 traits. The CPS probes are spaced at an average interval of 68 Kb. We have assessed the performance of this chip on 420 pepper germplasms, with successful capture of target DNA fragments by 45,387 probes. Furthermore, the probe capture ratio surpassed 70% in 410 of the 420 germplasms tested. Using this chip, we have efficiently genotyped 273 germplasms for spiciness levels and elucidated the genetic relationships among 410 pepper germplasms. Our results allowed for precise clustering of sister lines and C. chinense germplasms. In addition, through a GWAS for helical fruit shape, we identified three quantitative trait loci (QTLs): heli2.1, heli11.1, and heli11.2. Within the heli11.1 QTL, a gene encoding the tubulin alpha chain was identified, suggesting its potential role in the helical growth pattern of pepper fruits. In summary, the 45K pepper GBTS liquid-phase gene chip offers robust detection of polymorphic sites and is a promising tool for advancing research into pepper germplasm and the breeding of new pepper varieties.

Hairpin-Empowered Invasive Reaction Combined with Catalytic Hairpin Assembly Cascade Amplification for the Specific Detection of Single-Nucleotide Polymorphisms.

Single-nucleotide polymorphism (SNP) is widely used in the study of disease-related genes and in the genetic study of animal and plant strains. Therefore, SNP detection is crucial for biomedical diagnosis and treatment as well as for molecular design breeding of animals and plants. In this regard, this article describes a novel technique for detecting SNP using flap endonuclease 1 (FEN 1) as a specific recognition element and catalytic hairpin assembly (CHA) cascade reaction as a signal amplification strategy. The mutant target (MT) was hybridized with a biotin-modified upstream probe and hairpin-type downstream probe (DP) to form a specific three-base overlapping structure. Then, FEN 1 was employed for three-base overlapping structure-specific recognition, namely, the precise SNP site identification and the 5' flap of DP dissociation. After dissociation, the hybridized probes were magnetically separated by a streptavidin-biotin complex. Especially, the ability to establish such a hairpin-type DP provided a powerful tool that could be used to hide the cut sequence (CS) and avoid false-positive signals. The cleaved CS initiated the CHA reaction and allowed superior fluorescence signal generation. Owing to the high specificity of FEN 1 for single base recognition, only the MT could be distinguished from the wild-type target and mismatched DNA. Owing to the dual signal amplification, as low as 0.36 fM MT and 1% mutation abundance from the mixtures could be detected, respectively. Furthermore, it could accurately identify SNPs from human cancer cells, as well as soybean leaf genome extracts. This strategy paves the way for the development of more precise and sensitive tools for diagnosing early onset diseases as well as molecular design breeding tools.

Rapid detection of the irinotecan-related UGT1A1 & 5-fluorouracil related DPYD polymorphism by asymmetric polymerase chain reaction melting curve analysis

BackgroundDetermination of DPYD and UGT1A1 polymorphisms prior to 5-fluorouracil and irinotecan therapy is crucial for avoiding severe adverse drug effects. Hence, there is a pressing need for accurate and reliable genotyping methods for the most common DPYD and UGT1A1 polymorphisms. In this study, we introduce a novel polymerase chain reaction (PCR) melting curve analysis method for discriminating DPYD c.1236G > A, c.1679 T > G, c.2846A > T, IVS14 + 1G > A and UGT1A1*1, *28, *6 (G71R) genotypes. MethodsFollowing protocol optimization, this technique was employed to genotype 28 patients, recruited between March 2023 and October 2023, at the First Affiliated Hospital of Xiamen University. These patients included 20 with UGT1A1 *1/*1, 8 with UGT1A1 *1/*28, 4 with UGT1A1 *28/*28, 22 with UGT1A1*6 G/G, 6 with UGT1A1*6 G/A, 4 with UGT1A1*6 A/A, 27 with DPYD(c.1236) G/G, 3 with DPYD(c.1236) G/A, 2 with DPYD(c.1236) A/A, 27 with DPYD(c.1679) T/T, 2 with DPYD(c.1679) T/G, 3 with DPYD(c.1679) G/G, 28 with DPYD(c.2846A/T) A/A, 2 with DPYD(c.2846A/T) A/T, 2 with DPYD(c.2846A/T) T/T, 28 with DPYD(c.IVS14 + 1) G/G, 2 with DPYD(c.IVS14 + 1) G/G, and 2 with DPYD(c.IVS14 + 1) G/G, as well as 3 plasmid standards. Method accuracy was assessed by comparing results with those from Sanger sequencing or Multiplex quantitative PCR(qPCR). Intra- and inter-run precision of melting temperatures (Tms) were calculated to evaluate reliability, and sensitivity was assessed through limit of detection examination. ResultsThe new method accurately identified all genotypes and exhibited higher accuracy than Multiplex qPCR. Intra- and inter-run coefficients of variation for Tms were both ≤1.97 %, with standard deviations ≤0.95 °C. The limit of detection was 0.09 ng/μL of input genomic DNA. ConclusionOur developed PCR melting curve analysis offers accurate, reliable, rapid, simple, and cost-effective detection of DPYD and UGT1A1 polymorphisms. Its application can be easily extended to clinical laboratories equipped with a fluorescent PCR platform.