Detection Of Single Nucleotide Polymorphisms Research Articles

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.

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.

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 of genotype imputation from low-coverage whole-genome sequencing in scallops

Advancements in sequencing technologies have facilitated the use of low-coverage whole-genome sequencing (lcWGS) for detecting millions of single nucleotide polymorphisms (SNPs) in large populations at a low cost. The effectiveness of this approach largely relies on genotype imputation following lcWGS. While several imputation methods have been applied in human and other terrestrial animals, their performance has yet to be fully assessed in aquaculture organism, especially molluscs species which contribute 70% mariculture production in China and are characterized by high genetic polymorphism. This study aims to optimize reference panel construction and evaluate imputation strategies for SNP detection in two scallops, Chlamys farreri and Patinopecten yessoensis, both of which are important massively cultured bivalve species. High-coverage whole-genome sequencing (hcWGS) data are utilized to construct haplotype reference panels, and three imputation methods are applied: Beagle5.4, GLIMPSE2, and QUILT. The results indicated that QUILT outperformed other methods in scallop imputation, achieving imputation quality scores (IQS) exceeding 0.930 in C. farreri and 0.946 in P. yessoensis. Additionally, QUILT yielded the highest number of imputed loci in both species. Furthermore, the panel construction strategy was optimized by adjusting for genotype quality (GQ) and minor allele frequency (MAF). It is determined that a GQ threshold above 20 and a MAF threshold >0.01 are optimal. The effects of sequencing depth and sample size on imputation quality were also assessed, revealing that as sequencing depth increases, imputation quality improves, plateauing at 0.5×. Additionally, the impact of sample size varies depending on the imputation method. Moreover, negative correlation was found between imputation quality and the ratio of nonsynonymous to synonymous substitution rates (Ka/Ks) across chromosomal segments, highlighting the influence of selection pressure in the genome on imputation efficacy. This study provides an optimized comprehensive framework for SNP genotyping using lcWGS in scallops, with implications for aquaculture breeding and genetic research.

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.

Erratum: Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform.

This corrects the article 10.3791/66058.

Custom-Designed Probes for the Accurate Determination of Epidermal Growth Factor Receptor Mutations and Their Allelic Configuration.

The identification of single nucleotide polymorphisms (SNPs) is of paramount importance for disease diagnosis and clinical prognostication. In the context of nonsmall cell lung cancer (NSCLC), the emergence of resistance mutations, exemplified by the epidermal growth factor receptor (EGFR) T790 M and C797S, is intricately linked to the therapeutic efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Herein, a highly efficient and specific SNP detection platform for T790 M and C797S mutations has been engineered through the integration of an asymmetric polymerase chain reaction (PCR) and an ingeniously tailored four-way junction (4WJ) probe. Notably, a molecular beacon (MB) probe was judiciously designed to discern the allelic configuration of these mutations. The administration of first- and third-generation EGFR-TKIs demonstrates therapeutic efficacy solely when the mutations are in the trans configuration, characterized by a low fluorescence signal. In contrast, significant fluorescence by the MB probe is indicative of the C797S mutation being in a cis arrangement with T790M, thereby rendering the cells refractory to the therapeutic interventions of both first- and third-generation EGFR-TKIs. The assay is capable of concurrently detecting two point-mutations and ascertaining their allelic positions in a single test within 1.5 h, enhancing both efficiency and simplicity. It also exhibits high accuracy in the identification of clinical samples, offering promising implications for therapeutic guidelines. By enabling tailored treatment plans based on specific genetic profiles, our approach not only advances the precision of NSCLC treatment strategies but also marks a significant contribution to personalized medicine.

A multiplex SNaPshot assay for the detection of single nucleotide polymorphism associated with clinical severity of enterovirus infections

Non-polio enterovirus infections are known to cause a variety of diseases and neurological complications. It is also known that the severity of these diseases largely differs among individuals with different genotypes and alleles. The Single Nucleotide Polymorphisms (SNPs) within specific genes have a considerable effect on the immune response to enteroviruses and on the outcome of disease, leading to variations in complications and infection susceptibility. Knowing the distribution of such SNPs can be valuable for individual case management and studying epidemiological parameters of enterovirus infections. In this feasibility study, a multiplex version of the primer extension-based technique called the SNaPshot Assay has been developed to examine SNPs in various relevant genes for predicting the clinical severity of enterovirus infections. It is already established that this technique is precise, consistent, scalable, and likely to exhibit high throughput. The multiplex SNaPshot can investigate multiple genetic susceptibility markers simultaneously, and the assay can be used to identify vulnerable populations, understand the epidemiology of infections, and manage the outbreaks of enteroviruses. Based on the literature, 15 SNPs were identified which are suspected for higher susceptibility to the worst outcomes after enterovirus infection and the assay was developed. Blood samples of 100 healthy volunteers were collected and tested for assay feasibility as well as to know the proportions of 15 selected SNPs. After the analysis, seven SNPs have been identified and suggested to be considered for future assays. Based on the pilot test results, it appears that positivity for any three out of the identified seven SNPs might indicate a higher risk, and future studies correlated with clinical studies among patients with and without severe diseases utilizing this assay will provide robust parameters to determine at-risk individuals more accurately.

#1861 Association of the polymorphisms of the angiotensin converting enzyme and FRMD3 genes in the development of diabetic kidney disease

Abstract Background and Aims Individual genetic biomarkers, called single nucleotide polymorphisms (SNPs), have been proposed that could represent potential non-invasive markers for the early diagnosis of diabetic kidney disease (DKD), in addition to their association with a characteristic phenotype. Aim To determine the association of polymorphisms of the Angiotensin Converting Enzyme and FRMD3 genes with the risk of developing ERD in patients with type 2 diabetes. Method Case-control study, comparative, observational, ambispective and cross-sectional, in which men and women, over 18 years of age with T2D with and without ERD and healthy patients, who underwent DNA extraction, participated. genomic and subsequent genotyping, and detection of SNPs by mass spectrometry. Results A total of 400 patients participated, 199 men and 201 women, within which they were divided into three groups: 100 patients with type 2 diabetes and ERD confirmed by renal biopsy, 100 patients with type 2 diabetes without ERD and 200 healthy controls. A total of 3 polymorphisms of the ACE and FRMD3 genes were studied. Among the polymorphisms analyzed in the logistic regression, rs179975 CA (OR, 6.2; 95% CI: 1.15-7.48, p = 0.03), rs1888747 GA (OR, 6.6; 95% CI: 4.60-8.45, p = 0.002) and rs10868025 resulted. CG (OR, 4.1; 95% CI: 2.40-5.60, p = 0.002) Table 1. Conclusion Our study demonstrated the association of well-established genetic variants of the ACE and FRMD3 gene polymorphisms with the presence of ERD, showing that these SNPs, specifically rs179975 CA, rs1888747 GA and rs10868025 GC, may represent important genetic markers in the development of ERD and could be a predisposing factor due to the high frequency and strength of association that were observed in our studied population, this being the first study in Latin America.

Double base mismatches mediated catalytic hairpin assembly for enzyme-free single-base mutation detection: integrating signal recognition and amplification in one.

Single nucleotide polymorphism (SNP) biosensors are emerging rapidly for their promising applications in human disease prevention diagnosis, treatment, and prognosis. However, it remains a bottleneck in equipping simple and stable biosensors with the traits of high sensitivity, non-enzyme, and low cost. Double base mismatches mediated chain displacement reactions have attracted fascinating advantages of tailorable thermodynamics stability, non-enzyme, and excellent assembly compliance to involvement in SNP identification. As the base mismatch position and amount in DNA sequence can be artificially adjusted, it provides plenty of selectivity and specificity for exploring perfect biosensors. Herein, abiosensor with double base mismatches mediated catalytic hairpin assembly (CHA) is designed via one base mismatch in the toehold domain and the other base mismatch in the stem sequence of hairpin 1 (H1) by triggering CHA reaction to achieve selective amplification of the mutation target (MT) and fluorescence resonance energy transfer (FRET) effect that is composed of Cy3 and Cy5 terminally attached H1 and hairpin 2 (H2). Depending on the rationally designed base mismatch position and toehold length, the fabricated biosensors show superior SNP detection performance, exhibiting a good linearity with high sensitivity of 6.6 fM detection limitand a broad detection abundance of 1%. The proposed biosensor can be used to detect the KRAS mutation gene in real samples and obtain good recoveries between 106 and 116.99%. Remarkably, these extendible designs of base mismatches can be used for more types of SNP detection, providing flexible adjustment based on base mismatch position and toehold length variations, especially for their thermodynamic model for DNA-strand displacement reactions.

A novel high sensitive, specificity duplex enzyme-activated differentiating probes PCR method for the SNP detection and differentiation of MS-H vaccine strains from wild-type Mycoplasma synoviae strains

Mycoplasma synoviae (MS) is a contagious pathogen that poses a significant threat to the poultry industry. Detection plays an important role in the prevention and control of MS, particularly in differentiating between wild-type MS and live attenuated vaccine strains for vaccination selection and culling of animals with wild-type only. The live attenuated ts+ vaccine strain MS-H is recognized as the most effective and widely used vaccine. In this study, we have developed a method called double enzyme-activated differentiation probes PCR (DEA-probes PCR) for the differentiation of MS-H vaccine strain from wild-type strain by targeting the single nucleotide polymorphism (SNP) of the 367th nucleotide in the Obg gene sequence. We developed 2 modified probes with the ribonucleotide insert. When the probe perfectly complements with the target, the ribonuclease H2 (RNase H2) will cleave the ribonucleotide, resulting in the generation of fluorescent signal. With a detection limit of 5.8 copies/µL, the DEA-probes PCR method demonstrates 100% specificity in distinguishing wild-type MS from MS-H strains in 1 h. The method demonstrated great performance in real application of 100 superior palate cleft swab samples from chickens in poultry farms. Twenty-eight samples were detected as MS positive, consistent with the results of the Chinese industry standard method. Additionally, our method was able to distinguish 19 wild-type MS strains from 9 MS-H vaccine strains. The DEA-probes PCR method is rapid, specific and sensitive for SNP detection, overcoming the misidentification in MS detection and differentiation. It can be also applied to the differentiation of infected from vaccinated animals (DIVA) for other pathogens.

Multi-Gene Single Nucleotide Polymorphism Detection in Gastric Cancer Based on Ion Semiconductor Sequencing Platform.

Gastric cancer is a common heterogeneous tumor. Most patients have advanced gastric cancer at the time of diagnosis and often need chemotherapy. Although 5-fluorouracil (5-FU) is widely used for treatment, its therapeutic sensitivity and drug tolerance still need to be determined, which emphasizes the importance of individualized administration. Pharmacogenetics can guide the clinical implementation of individualized treatment. Single nucleotide polymorphisms (SNPs), as a genetic marker, contribute to the selection of appropriate chemotherapy regimens and dosages. Some SNPs are associated with folate metabolism, the therapeutic target of 5-FU. Methylenetetrahydrofolate reductase (MTHFR) rs1801131 and rs1801133, dihydrofolate reductase (DHFR) rs1650697 and rs442767, methionine synthase (MTR) rs1805087, gamma-glutamyl hydrolase (GGH) rs11545078 and solute carrier family 19 member 1 (SLC19A1) rs1051298 have been investigated in different kinds of cancers and antifolate antitumor drugs, which have potential forecasting and guiding significance for application of 5-FU. The ion torrent next-generation semiconductor sequencing technology can rapidly detect gastric cancer-related SNPs. Each time a base is extended in a DNA chain, an H+ will be released, causing local pH changes. The ionic sensor detects pH changes and converts chemical signals into digital signals, achieving sequencing by synthesis. This technique has low sample requirement, simple operation, low cost, and fast sequencing speed, which is beneficial for guiding individualized chemotherapy by SNPs.

Long-term toxicity evaluation of aristolochic acid-IIIa in mice

Aristolochic acid (AA)-IIIa is an AA analog present in Aristolochiaceae plants. To evaluate the chronic toxicity of AA-IIIa, mice were intragastrically administered with media control, 1 mg/kg AA-IIIa, and 10 mg/kg AA-IIIa, and designated as the control (CTL), AA-IIIa low dose (AA-IIIa-L), and AA-IIIa high dose (AA-IIIa-H) groups, respectively. AA-IIIa was administered three times a week, every other day, for 24 weeks (24-week time point). Thereafter, some mice were sacrificed immediately, while others were sacrificed 29 or 50 weeks after AA-IIIa withdrawal (53- or 74-week time point). Serum and organs were collected for biochemical and pathological analyses, respectively. Whole-genome sequencing was performed on the kidney, liver, and stomach tissues of AA-IIIa-treated mice for single-nucleotide polymorphism (SNP) detection. AA-IIIa-H mice died at 66 weeks, and the remaining mice showed moribund conditions at the 69 weeks. AA-IIIa induced minor kidney tubule injury, fibroblast hyperplasia, and forestomach carcinoma in mice. Bladder, intestine, liver, heart, spleen, lung, and testis tissues were not pathologically altered by AA-IIIa. In addition, AA-IIIa increased the C:G > A:T mutation in the kidney; however, no SNP mutation changes were observed in the liver and forestomach tissues of AA-IIIa-H mice at the 24-week time point compared with control mice. Therefore, we suspect that AA-IIIa is potentially mutagenic for mice after overdose and long-term administration. On the other hand, the forestomach is a unique organ in mice, but it does not exist in humans; thus, we hypothesize that the stomach toxicity induced by AA-IIIa is not a suitable reference for toxicological evaluation in humans. We recommend that Aristolochiaceae plants containing AA-IIIa should be properly supervised, and overdosing and long-term administration of drugs containing AA-IIIa should be avoided.

In situ quantitative mapping of coding single nucleotide polymorphism on mRNA inside cells by SERS-fluorescence dual-mode probe

Surface Enhanced Raman Scattering (SERS) has potential in nucleic acid detection, but its ability on single nucleotide polymorphisms (SNPs) detection in the early diagnosis of diseases is still limited. In fact, there is few research about in situ detection of coding SNPs which are carried on mRNA. In this paper, we developed SERS-Fluorescence Dual-mode probes to achieve in situ quantitative mapping of intracellular cSNP. The probes were based on Au@Ag core-shell structure that possessed both excellent biocompatibility and localized surface plasmon resonance. Fluorescence can provide a rapid visualization of intracellular cSNP, while SERS can achieve its in situ quantification. The quantitation was conducted by comparing the SERS signals of the reporting molecule p-mercaptobenzoic acid and fluorescent molecule FAM, the detecting limitation of which can be achieved as low as 1.4 fM. This ratiometric detection is more accurate than traditional analysis based on signal intensity directly. It successfully achieved intracellular imaging of the G12D mutation site of KRAS gene in A549 cells, which can help to get the distribution of cSNP in single cell more clearly. And it also demonstrated the good mapping ability for biomolecules (1003 cm−1 for phenylalanine) simultaneously. In conclusion, this kind of probe could be used as a potential tool to monitor cSNP as well as biomolecules in multiple biological processes, which could provide reference for disease prediction.