Detection Of Single Nucleotide Polymorphisms Research Articles

Benzimidazole Resistance-Associated Mutations in the β-tubulin Gene of Hookworms: A Systematic Review.

There is a growing number of reports on the occurrence of benzimidazole resistance-associated single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene of various helminths of veterinary, and public health concerns. However, a comprehensive analysis of their occurrence, and their contributions to conferring benzimidazole resistance among hookworms has yet to be done. The objectives of this systematic review are to summarize and synthesize peer-reviewed evidence on the occurrence of these resistance-associated mutations in hookworms, document their geographical distribution, and assess their contributions to conferring phenotypic resistance. Three databases were systematically searched using specific keywords. Research that assessed the occurrence of benzimidazole resistance-associated SNPs in hookworms, papers that reported the geographical distribution of these SNPs, and studies that investigated the SNPs' resistance-associated phenotypic effects were included in the review. Research that was not done in hookworms, papers not in the English language, and literature reviews and book chapters were excluded. Critical appraisal checklists were used to determine the risk of bias in the selected papers. Data were extracted from the selected studies and analyzed. PROSPERO Systematic Review Protocol Registration No.: CRD42024510924. A total of 29 studies were included and analyzed. Of these, four were conducted in a laboratory setting, eight described the development and validation of SNP detection methods, and the remaining 17 involved field research. Seven SNP-induced amino acid substitutions at four loci were reported among several hookworm species: Q134H, F167Y, E198A, E198K, E198V, F200Y, and F200L. SNPs have been reported in isolates occurring in the United States, Canada, Brazil, Haiti, Australia, New Zealand, Kenya, Ghana, Mozambique, and Tanzania. Resistance mutations have not been reported in Asia. E198A and F200L were reported in Ancylostoma ceylanicum with laboratory-induced resistance. F167Y and Q134H conferred resistance in A. caninum, as revealed by in vitro investigations and field assessments. There is insufficient peer-reviewed evidence to prove the association between SNP occurrence and resistance. Mutations in the β-tubulin isotype 1 gene confer benzimidazole resistance in A. caninum and A. ceylanicum, but similar evidence is lacking for other human hookworms. Understanding benzimidazole resistance through further research can better inform treatment, prevention, and control strategies.

Newly developed QCM-DNA biosensor for SNP detection in small DNA fragments: A wine authenticity case study

We propose a QCM-DNA biosensor for single nucleotide polymorphism (SNP) detection in samples of differing complexity. An optimized protocol is presented, focusing on parameters affecting probe immobilization and hybridization efficiency. Our results led to the implementation of thiolated probe reduction with TCEP, followed by immobilization in PBS buffer containing MgCl2. The biosensor exhibited an enhanced specificity at 37 °C, achieving detection of single mismatches using synthetic targets. Using real samples, we applied the biosensor in a wine authenticity assessment context. The addition of dithiothreitol improved stability and reproducibility when testing wine DNA samples. The QCM-DNA biosensor was able to specifically detect complementary DNA in leaf and wine DNA samples, distinguishing between samples with two heterozygous mismatches. The biosensor solely depends on DNA extraction, basic instrumentation, and reagents, without requiring PCR or signal amplification strategies. Our findings show the biosensor potential for applications in wine authenticity assessment and other fields requiring complex analysis in undemanding settings.

Optimization of Whole-Genome Resequencing Depth for High-Throughput SNP Genotyping in Litopenaeus vannamei.

The application of whole-genome resequencing in genetic research is rapidly expanding, yet the impact of sequencing depth on data quality and variant detection remains unclear, particularly in aquaculture species. This study re-sequenced 31 Litopenaeus vannamei (L. vannamei) samples at over 28× sequencing depth using the Illumina NovaSeq system and down-sampled the data to simulate depths from 0.5× to 20×. Results showed that when the sequencing depth was below 10×, the number of SNP identifications increased sharply with the rise in depth, with single nucleotide polymorphisms (SNPs) detected at 10× accounting for approximately 69.16% of those detected at 20×. The genotyping accuracy followed a similar trend to SNP detection results, being approximately 0.90 at 6×. Further analyses showed that the main cause of genotyping errors was the misidentification of heterozygous variants as homozygous variants. Therefore, considering both the quantity and quality of SNPs, a sequencing depth of 10× is recommended for whole-genome studies and genetic mapping, while a depth of 6× is more cost-effective for population structure analysis. This study underscores the importance of selecting optimal sequencing depth to ensure reliable variant detection and high data quality, providing valuable guidance for whole-genome resequencing in shrimp and other aquatic species.

Fluorescent primers amplification refractory mutation system qPCR (FP ARMS-qPCR) for MTHFR C677T SNP genotyping.

The currently used methods for the detection of methylene tetrahydrofolic acid reductase (MTHFR) C677T single nucleotide polymorphism (SNP) are either time-consuming or expensive. In this study, we devised an accurate, rapid and easy-to-use SNP detection system based on fluorescent primers amplification refractory mutation system qPCR, known as FP ARMS-qPCR. Fluorescent primers (FPs) modified by fluorescent dye or quencher near the 3' terminal thymine were designed. The reaction conditions were optimized and the performance was evaluated. Using commercial kits as controls, 242 samples were tested in parallel to verify the feasibility of the FP ARMS-qPCR assay. We demonstrated the good sensitivity and specificity of the FP ARMS-qPCR with optimized conditions. The assay was able to accurately distinguish between different SNP sites of MTHFR C677T in less than 2h using as low as 50 pg of template genomic DNA. Completely consistent genotyping results reveal that FP ARMS-qPCR is concordant with commercial kits. We established a specific, sensitive, and rapid FP ARMS-qPCR method for the detection of MTHFR C677T genotype. This could also serve as a potential diagnostic tool for a variety of diseases.

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.

Genetic polymorphism of IL-40 in male patients with ankylosing spondylitis

Background: Ankylosing spondylitis (AS) is a kind of arthritis marked by persistent inflammation in the spine's joints, usually at the point where the spine and pelvis meet. Such a disease has different causes, including immunological causes. Therefore, it is necessary to investigate the role of the immune system in this disease to improve a potential treatment strategy. In this context, the present study examined the single nucleotide polymorphisms (SNPs) of the IL-40 gene of IL-40 in a group of Iraqi males with AS to determine their associations in AS pathogenesis. Method: The study included a total of 200 Iraqi male participants (100 with AS and 100 healthy subjects). The detection of SNPs of IL-40 was performed using polymerase chain reaction (PCR) techniques followed by the Sanger sequencing method. Multinomial logistic regression analysis was used to analyze the SNPs under five genetic models (allele, recessive, dominant, and co-dominant). Results: Detected that the heterozygous genotype (GA and AA) and alleles (G and A) of rs72896265, the heterozygous genotype (AG and GG) and alleles (A and G) of rs60316778 and both heterozygous genotype (CC) and alleles (C and T) of rs9915090 were significantly associated with AS risk (p < 0.001).

Development of a Spot Test (ST) Based on the Nucleic Acid Amplification Test (NAAT) for Ginseng Species Authentication

It is very challenging to distinguish between Panax ginseng (P. ginseng) and Panax quinquefolius (P. quinquefolius) based on their physical appearances. Therefore, a molecular test that can be performed in commercial settings is needed to address this challenge. We have selected a locus containing a single nucleotide polymorphism (SNP) site in the Dammarendiol-II Synthase (DS) gene to differentiate between P. ginseng and P. quinquefolius. An isothermal nucleic acid amplification test (NAAT) developed previously was adapted and used on a spot test (ST) format consisting of a sample pad, a nitrocellulose membrane, and an adsorbent pad. An ST with a colorimetric detection method has been developed with visual detection based on 20-nm gold nanoparticles. The presence of the target DNA will generate a red color signal, which is visible to the naked eye. The assay comprises the capture probes, constructed based on the DS gene of ginseng, and the ends of the probe sequences sit on the SNP site for the P. ginseng and P. quinquefolius sequences; the detection probe employed in the assay is tagged with gold. The NAAT amplifies the extracted plant genomic samples and enhances the detection of specific SNPs. This NAAT technique was used to authenticate two ginseng root samples, showing greater specificity ratios than our previous work.

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.

TLR4 and TNF-α single nucleotide polymorphisms in patients with brucellosis: Association with infection complications

ObjectivesTo investigate associations of the carriage of single nucleotide polymorphisms (SNPs) of proteins involved in the immune response of patients with brucellosis. MethodsA case control study of patients with brucellosis upon WHO criteria. Blood genomic analysis was performed by RFLP- PCR for the detection of SNPs: i) at promoters -376 G > A (rs1800750); -308 G > A (rs 1,800,629); -238 G > A (rs361525) of the TNF gene, ii) at -896 A > G Asp299Gly (rs4986790) and -1196 C > T Thr399Ile (rs4986791) positions of the TLR-4 gene. Logistic regression analysis of factors related to brucellar spondylodiscitis was performed. ResultsPatients with brucellosis (n = 105) were male (n = 67, 63.8 %); mean age (SD): 49.51(18.31); spondylodiscitis (n = 30), sacral osteomyelitis (n = 21). Carriage of the minor frequency A alleles at -238 of the promoter region of TNF was greater in patients than in controls (11.4% vs 2.6 %, p < 0.001). In a stepwise regression model including host variables and TNF-238 G A-1 genotype, only the last one was associated with brucellar spondylodiscitis [OR 2.91 (CI95 % 1.02–8.31), p = 0.047]. ConclusionsIn our cohort, the association of one TNF SNP of patients with brucellosis, in particular spondylodiscitis, might be prognostic whereas further investigation of the exact role in the host immune response is required.

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.

Noninvasive Prenatal Genetic Screening of Cell-Free Fetal DNA for Early Prediction of β-Thalassemia Using Fiber Optic Nanogold-Linked Sorbent Assay.

β-Thalassemia is a prevalent type of severe inherited chronic anemia, primarily identified in developing countries. The identification of single nucleotide polymorphisms (SNPs) plays a vital role in the early diagnosis of genetic diseases. Here, we reported the development of an amplification-free fiber optic nanogold-linked sorbent assay method using a fiber optic particle plasmon resonance (FOPPR) biosensor for rapid and ultrasensitive detection of SNPs. Herein, MutS protein was selected as the biorecognition capture probe and immobilized on the sensing region to capture the target mutant DNA, which was hybridized with a single-base mismatched single-stranded DNA labeled by a gold nanoparticle (AuNP). The AuNP acts as a signaling agent to be detected by the FOPPR biosensor when it is bound on the fiber core surface. The method effectively differentiates mismatched double-stranded DNA by MutS protein from perfectly matched/complementary dsDNA. It exhibits an impressively low detection limit for the detection of SNPs at approximately 10-16 M using low-cost sensor chips and devices. By determination of the ratio of mutant DNA to normal DNA in cell-free genomic DNA from blood samples, this method is promising for diagnosing β-thalassemia in fetuses without invasive testing techniques.

Optimizing Strategy for Whole-Genome Genotype Imputation in Scallops

Advancements in sequencing technologies have facilitated low-coverage whole-genome sequencing (lcWGS) for detecting millions of single nucleotide polymorphisms (SNPs) in large populations at low cost. The effectiveness of this approach relies on genotype imputation following lcWGS. While several imputation methods have been applied in human and other terrestrial animals, their performance in aquaculture organisms, particularly mollusks, remains unassessed. Mollusks contribute 70% of mariculture production in China and are characterized by high genetic polymorphism. In this study, we aimed to optimize reference panel construction and evaluate imputation strategies for SNP detection in two scallops, Chlamys farreri and Patinopecten yessoensis, both important bivalve species. High-coverage whole-genome sequencing (hcWGS) data were used to construct haplotype reference panels, and three imputation methods were applied: Beagle5.4, GLIMPSE2, and QUILT. QUILT outperformed other methods, achieving imputation quality scores (IQS) exceeding 0.930 in C. farreri and 0.946 in P. yessoensis, and yielded the highest number of imputed loci in both species. Furthermore, panel construction was optimized by adjusting for genotype quality (GQ) and minor allele frequency (MAF); we determined that a GQ threshold > 20 and an MAF threshold > 0.01 were optimal. We observed that imputation quality improved with increased sequencing depth, plateauing at 0.5×, and varied with sample size depending on the imputation method. A negative correlation was observed between imputation quality and the ratio of non-synonymous to synonymous substitution rates (Ka/Ks) across chromosomal segments, highlighting the influence of selection pressure on imputation efficacy. Collectively, our findings provide an optimized framework for SNP genotyping using lcWGS in scallops, with implications for aquaculture breeding and genetic research.

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.

An enzyme-free fluorescence sensing platform based on multiplex toehold-mediated strand displacement for point-of-care testing of single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs) are biomarkers for the prediction and diagnosis of biomedical diseases, as well as important reference markers for intelligent design and breeding of animals and plants. Therefore, the development of high-sensitivity, practicability, and convenient SNPs sensing strategies and detection platforms is of great significance. In this study, we pioneered an ultra-sensitive universal sensing strategy for single base mutation detection and an enzyme-free FRET fluorescence sensing point-of-care testing (POCT) platform for SNP detection. Based on the thermodynamically regulated strand replacement mechanism, the system rationally introduced extra base mismatches and combined multiple toehold-mediated strand displacement (TMSD) reactions to achieve enzyme-free recognition of the single base mutation and double amplification of the detection signal. Finally, the biological signal of the mutant target (MT) was recognized, amplified, and transduced into a fluorescence resonance energy transfer (FRET) signal between Cy3 and Cy5 for output. Overall, the proposed biosensing strategy demonstrated high sensitivity for MT, with a detection limit as low as 0.37 fM, and could achieve detection of targets with mutant abundances as low as 0.1 %. Moreover, a portable detection system was assembled in conjunction with this sensing strategy to form a POCT platform for SNP genotyping. This POCT platform was validated to accurately identify SNPs from human cancer cells and soybean leaf genome extracts. In summary, this study will provide new insights into the design of enzyme-free biosensors for SNP detection, and will provide important technical support tools for the prevention and diagnosis of major diseases, animal and plant breeding, etc.

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.

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.

Development of DNA-Based Lateral Flow Assay for Detection of LDLR Gene Mutation for Familial Hypercholesterolemia.

The techniques for detecting single nucleotide polymorphisms (SNP) require lengthy and complex experimental procedures and expensive instruments that may only be available in some laboratories. Thus, a deoxyribonucleic acid (DNA)-based lateral flow assay (LFA) was developed as a point-of-care test (POCT) diagnostic tool for genotyping. In this study, single nucleotide variation (E101K) in the low-density lipoprotein receptor (LDLR) gene leading to familial hypercholesterolemia (FH) was chosen as a model. Hypercholesterolemic individuals (n = 103) were selected from the Malaysian Cohort project (UKM Medical Molecular Biology Institute) while the control samples were selected from the Biobank (UKM Medical Molecular Biology Institute). The DNA samples were isolated from whole blood. Polymerase chain reaction (PCR) amplification process was performed using bifunctional labelled primers specifically designed to correspond to the variant that differentiates wild-type and mutant DNA for visual detection on LFA. The variant was confirmed using Sanger sequencing, and the sensitivity and specificity of the LFA detection method were validated using the Agena MassARRAY® technique. Out of 103 hypercholesterolemic individuals, 5 individuals (4.8%) tested positive for E101K, LDLR mutation and the rest, including healthy control individuals, tested negative. This result was concordant with Sanger sequencing and Agena MassARRAY®. These five individuals could be classified as Definite FH, as the DNA diagnosis was confirmed. The sensitivity and specificity of the variant detection by LFA is 100% compared to results using the genotyping method using Agena MassARRAY®. The developed LFA can potentially be used in the POC setting for detecting the E101K variant in the LDLR gene. This LFA can also be used to screen family members with E101K variant in the LDLR gene and is applicable for other SNP's detection.

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.