Detection Of Single Nucleotide Polymorphisms Research Articles

Detection of SNPs in FABP4 Gene and Its Relationship with Milk Quality Traits in Iraqi Jenoubi Cows

Jenoubi cattle breed classified as a Bos indicus L., is one of Iraqi indigenous cattle that kept for milk production purpose, but the influence of fatty acid-binding protein 4 (FABP4) on milk-related traits remain poorly understood. The current study was conducted to investigate the effect of single nucleotide polymorphisms (SNPs) in the FABP4 gene of Jenoubi cows. The DNA samples were extracted from the blood of 21 cows. Along 565-bp nucleotide (nt) amplicon from the intron 2- exon 3- intron 3 of FABP4 was amplified by polymerase chain reaction (PCR) while forward sequencing was used to detect SNPs and results were analyzed using bioinformatics software. The findings resulted the existence of two SNPs (c.3689G>A and c.3709G>C) in the exon 3 and four SNPs (g.3494A>C, g.3531A>T, g.3743T>C and g.3765T>C) in the intron regions. Therefore, three haplotypes of FABP4 gene were obtained in this study based on three SNPs of g.3494A>C, g.3531A>T and g.3765T>C. Nonetheless, those haplotypes were not significantly influencing to the milk quality traits of Jenoubi cows. However, the SNPs examined in this study might not be used as potential DNA marker to improve milk production traits of the current breed.

Detection of SNPs and benzimidazole resistance in strongyle nematode eggs of horses by allele-specific PCR.

This study was conducted to determine single nucleotide polymorphisms (SNPs) and the benzimidazole (BZ) resistance in strongyle nematode egg populations in horses using molecular techniques. A total of 200 fecal samples were collected from horses in 26 farms in two provinces (Kayseri and Nevşehir) of the Central Anatolia Region of Türkiye between May and August 2022. The flotation method was used to detect strongyle nematode eggs in the fecal samples of the horses. Afterward, strongyle nematode eggs were collected, and the allele-specific polymerase chain reaction (AS-PCR) technique was used to detect the BZ resistance. BZ-susceptible and BZ-resistant PCR products were sequenced to determine single nucleotide polymorphisms (SNPs) in the β-tubulin isotype 1 gene. The strongyle nematode eggs were determined in 85 (42.5%) out of 200 fecal samples. AS-PCR detected 50.58% (43/85) BZ-resistant (homozygous resistant) and 36.4% (31/85) BZ-susceptible (homozygous susceptible) genes in the strongyle eggs. Both BZ-resistant and BZ-susceptible genes (heterozygous) were determined in 11 samples. BZ-resistant and BZ-susceptible allele frequencies were determined as 57.0% (48.5/85) and 43.0% (36.5/85), respectively. SNPs were detected only in codon 200 of the β-tubulin isotype 1 gene in four sequenced isolates of the two resistant and two susceptible isolates. This study is the first molecular report on BZ resistance in strongyle nematode eggs in horses in Türkiye. The widespread prevalence of BZ-resistant alleles in equine strongyle nematodes shows the requirement for the immediate usage of other anthelmintics instead of the BZ group drugs for the effective management and control of equine strongyle nematodes.

Comparison of Brucella abortus population structure based on genotyping methods with different levels of resolution

Numerous genotyping techniques based on different principles and with different costs and levels of resolution are currently available for understanding the transmission dynamics of brucellosis worldwide. We aimed to compare the population structure of the genomes of 53 Brazilian Brucella abortus isolates using eight different genotyping methods: multiple-locus variable-number tandem-repeat analysis (MLVA8, MLVA11, MLVA16), multilocus sequence typing (MLST9, MLST21), core genome MLST (cgMLST) and two techniques based on single nucleotide polymorphism (SNP) detection (parSNP and NASP) from whole genomes. The strains were isolated from six different Brazilian states between 1977 and 2008 and had previously been analyzed using MLVA8, MLVA11, and MLVA16. Their whole genomes were sequenced, assembled, and subjected to MLST9 MLST21, cgMLST, and SNP analyses. All the genotypes were compared by hierarchical grouping method based on the average distances between the correlation matrices of each technique. MLST9 and MLST21 had the lowest level of resolution, both revealing only four genotypes. MLVA8, MLVA11, and MLVA16 had progressively increasing levels of resolution as more loci were analyzed, identifying 6, 16, and 44 genotypes, respectively. cgMLST showed the highest level of resolution, identifying 45 genotypes, followed by the SNP-based methods, both of which had 44 genotypes. In the assessed population, MLVA was more discriminatory than MLST and was easier and cheaper to perform. SNP techniques and cgMLST provided the highest levels of resolution and the results from the two methods were in close agreement. In conclusion, the choice of genotyping technique can strongly affect one's ability to make meaningful epidemiological conclusions but is dependent on available resources: while the VNTR based techniques are more indicated to high prevalence scenarios, the WGS methods are the ones with the best discriminative power and therefore recommended for outbreaks investigation.

Development of a three-colour digital PCR for early and quantitative detection of benzimidazole resistance-associated single nucleotide polymorphisms in Haemonchus contortus

Haemonchus contortus is the most pathogenic nematode in small ruminants and anthelmintic resistance (AR) hampers its efficient control. Early detection of AR status is required to reduce selection for AR and cannot be achieved using phenotypic tests. For benzimidazoles (BZs), the detection of AR-associated alleles characterised by single nucleotide polymorphisms (SNPs) in the isotype 1 β-tubulin gene allows early AR detection in strongyles. The F200Y, F167Y, E198A and E198L polymorphisms have been described in BZ-resistant populations with a clear variation in frequencies between regions. A novel digital PCR (dPCR) enables the detection of all of the above-described polymorphisms in H. contortus. Assays were validated using synthetic DNA fragments containing these SNPs. Then, larvae obtained and pooled at farm level from 26 Austrian and 10 Italian sheep farms were analysed. For all assays a detection limit of 15 copies/μl of resistance alleles and a high level of accuracy were demonstrated, allowing to detect allele frequencies of 1% in most samples. In Austrian samples, elevated frequencies of F200Y resistance alleles were detected on all farms. Polymorphisms in codon 167 and codon 198 were identified in H. contortus from Austria for the first time. In Italian samples, the frequency of resistance alleles was still comparatively low, but F200Y resistance alleles were traceable. In conclusion we developed for the first time dPCR assays that target all SNPs of relevance associated with BZ-resistance in H. contortus. Future research on AR development could benefit from an early onset of SNP-based surveillance that would include the developed assays for all SNPs of relevance. Improved surveillance in the long term will include other important, though less pathogenic, nematode genera in the analyses.

Integration of CRISPR/Cas13a and V-Shape PCR for Rapid, Sensitive, and Specific Genotyping of CYP2C19 Gene Polymorphisms.

Rapid detection of single nucleotide polymorphisms (SNPs) in the CYP2C19 gene is of great significance for clopidogrel-accurate medicine. CRISPR/Cas systems have been increasingly used in SNP detection due to their single-nucleotide mismatch specificity. PCR, as a powerful amplification tool, has been incorporated into the CRISPR/Cas system to improve the sensitivity. However, the complicated three-step temperature control of the conventional PCR impeded rapid detection. The "V" shape PCR can shorten about 2/3 of the amplification time compared with conventional PCR. Herein, we present a new system termed the "V" shape PCR-coupled CRISPR/Cas13a (denoted as VPC) system, achieving the rapid, sensitive, and specific genotyping of CYP2C19 gene polymorphisms. The wild- and mutant-type alleles in CYP2C19*2, CYP2C19*3, and CYP2C19*17 genes can be discriminated by using the rationally programmed crRNA. A limit of detection (LOD) of 102 copies/μL was obtained within 45 min. In addition, the clinical applicability was demonstrated by genotyping SNPs in CYP2C19*2, CYP2C19*3, and CYP2C19*17 genes from clinical blood samples and buccal swabs within 1 h. Finally, we conducted the HPV16 and HPV18 detections to validate the generality of the VPC strategy.

Variety Differentiation: Development of a CRISPR DETECTR Method for the Detection of Single Nucleotide Polymorphisms (SNPs) in Cacao (Theobroma cacao) and Almonds (Prunus dulcis)

To prevent food fraud, products can be monitored by various chemical-analytical techniques. In this study, we present a CRISPR-Cpf1 DETECTR-based assay for the differentiation of plant ingredients in sweet confectionary like fine and bulk-cocoa, or bitter and sweet almonds. To enable rapid in-field analysis, the trans-cleavage activity of the Cpf1 enzyme was used to develop a DETECTR (DNA endonuclease-targeted CRISPR trans reporter) assay for simple, highly specific fluorometric detection of single nucleotide polymorphisms (SNPs). The endonuclease Cpf1 requires the protospacer adjacent motif (PAM) 5’-TTTV-3’ for activation, but the recognition sequence is freely programmable. The SNPs were selected to alter the Cpf1 specific PAM sequence. As a result, sequences that do not carry the canonical PAM sequence are not detected and thus not cut. The optimized system was used for both raw material and processed products such as cocoa masses or marzipan with a limit of detection of 3 ng template DNA. In addition, we were able to implement the system in the context of an LFA (lateral flow assay) to serve as a basis for the development of rapid test systems.

Genome-wide detection of m6A-associated SNPs in atrial fibrillation pathogenesis.

N6-Methyladenosine (m6A) modification is of great importance in both the pathological conditions and physiological process. The m6A single nucleotide polymorphisms (SNPs) are associated with cardiovascular diseases including coronary artery disease, heart failure. However, it is unclear whether m6A-SNPs are involved in atrial fibrillation (AF). Here, we aimed to explore the relationship between m6A-SNPs and AF. The relationship between m6A-SNPs and AF was evaluated by analyzing the AF genome-wide association study (GWAS) and m6A-SNPs annotated by the m6AVar database. Further, eQTL and gene differential expression analysis were performed to confirm the association between these identified m6A-SNPs and their target genes in the development of AF. Moreover, we did the GO enrichment analysis to figure out the potential functions of these m6A-SNPs affected genes. Totally, 105 m6A-SNPs were identified to be significantly associated with AF (FDR < 0.05), among which 7 showed significant eQTL signals on local genes in the atrial appendage. By using four public AF gene expression datasets, we identified genes SYNE2, USP36, and THAP9 containing SNPs rs35648226, rs900349, and rs1047564 were differentially expressed in AF population. Further, SNPs rs35648226 and rs1047564 are potentially associated with AF by affecting m6A modification and both of them might have an interaction with RNA-binding protein, PABPC1. In summary, we identified m6A-SNPs associated with AF. Our study provided new insights into AF development as well as AF therapeutic target.

Simultaneous selection of multiple important single nucleotide polymorphisms in familial genome wide association studies data

We propose a resampling-based fast variable selection technique for detecting relevant single nucleotide polymorphisms (SNP) in a multi-marker mixed effect model. Due to computational complexity, current practice primarily involves testing the effect of one SNP at a time, commonly termed as ‘single SNP association analysis’. Joint modeling of genetic variants within a gene or pathway may have better power to detect associated genetic variants, especially the ones with weak effects. In this paper, we propose a computationally efficient model selection approach—based on the e-values framework—for single SNP detection in families while utilizing information on multiple SNPs simultaneously. To overcome computational bottleneck of traditional model selection methods, our method trains one single model, and utilizes a fast and scalable bootstrap procedure. We illustrate through numerical studies that our proposed method is more effective in detecting SNPs associated with a trait than either single-marker analysis using family data or model selection methods that ignore the familial dependency structure. Further, we perform gene-level analysis in Minnesota Center for Twin and Family Research (MCTFR) dataset using our method to detect several SNPs using this that have been implicated to be associated with alcohol consumption.

Length-Dependent Gold Nanoparticle-Labeled Nucleic Acid Probe Based Dual Test Strip for Visualized Discrimination of Single Nucleotide Polymorphism

Single nucleotide polymorphism (SNP) is closely related to the susceptibility of various common diseases and adverse drug reactions. Conclusively, SNP detection is significant for early-stage disease diagnosis and personalized medicine. The gold nanoparticle nucleic acid strip has the advantages of low-cost and rapid visualization (within 5–10 min), and it was introduced into detecting cancer-related SNP sites (A/A, A/G, G/G) in the CYP1A1 gene. As an efficient visualized approach based on the observation of the test zone (TZ) on the strip, it is improper to develop color at the TZ toward the negative sample; otherwise, it may lead to invalid false positive results. Thus, it is crucial to avert such a situation. In this work, dual strips named A and G were prepared, which were completely complementary to the T/T site (complementary base of A/A site) and C/C site (complementary base of G/G site), respectively. Single-strand DNA (ssDNA) containing SNP sites was obtained by conventional two-step polymerase chain reaction (PCR). Then SNP sites could be directly visualized and accurately distinguished on the dual strips. Furthermore, the false positive result could be eliminated by optimizing the length of DNA modified on gold nanoparticle and the concentration of NaCl. The result for the detection of 21 human blood samples with this method was consistent with those of sequencing. The platform exhibits the benefits of rapidity, easily visual determination, is false-positive-free, and is low-cost and plays an important part in the initial screening of SNP-related diseases.

Genome-wide association screening and MassARRAY for detection of high-temperature resistance-related SNPs and genes in a hybrid abalone (Haliotis discus hannai ♀ × H. fulgens ♂) based on super genotyping-by-sequencing

High seawater temperatures in summer often lead to high mortality in farmed abalone, which greatly increases the cost of farming. The hybrid abalone (Haliotis discus hannai ♀ × H. fulgens ♂, DF), is one of the main varieties of abalone cultured in Fujian Province, China, to resist the high temperature. In this study, the high-temperature resistance of DF abalones was assessed under 28 °C - 32 °C. 31 °C was applied in the subsequent screening experiments of high-temperature resistant individuals. A total of 614,321 single nucleotide polymorphisms (SNPs) were identified via super genotyping-by-sequencing (S-GBS), and 69 SNPs involved in 42 candidate genes were identified by genome-wide association study (GWAS). In addition, according to gene functional annotation, the locus with the lowest P value was screened within the threshold range (−log10P = 4.98296). The genotyping was verified by Sequenom MassARRAY, and 7 SNPs were proved to be related to high temperature resistance in DF population of 100 individuals. 7 candidate genes (fabp, e3rglg4, rgs2, tanc2x6, osbp1, arsb, and uch1) were identified near these SNPs. Finally, these candidate genes were further proved to be associated with the high-temperature resistance trait of DF by the analysis of differential expression genes between sensitive and tolerant individuals. These candidate genes involved regulatory mechanisms such as cell stability, protein repair, lipid metabolism and immune regulation. Notably, the mechanism by which the e3rglg4 gene was involved in the degradation of abnormally folded proteins of abalone under heat stress, corroborating the reliability of the results of GWAS and Sequenom MassARRA. These results complement the shortage of molecular markers for heat resistance of hybrid abalone, provide a reference for marker-assisted selection (MAS) and accelerate the development of heat-resistant abalone varieties.

Combining GS-assisted GWAS and transcriptome analysis to mine candidate genes for nitrogen utilization efficiency in Populus cathayana

BackgroundForest trees such as poplar, shrub willow, et al. are essential natural resources for sustainable and renewable energy production, and their wood can reduce dependence on fossil fuels and reduce environmental pollution. However, the productivity of forest trees is often limited by the availability of nitrogen (N), improving nitrogen use efficiency (NUE) is an important way to address it. Currently, NUE genetic resources are scarce in forest tree research, and more genetic resources are urgently needed.ResultsHere, we performed genome-wide association studies (GWAS) using the mixed linear model (MLM) to identify genetic loci regulating growth traits in Populus cathayana at two N levels, and attempted to enhance the signal strength of single nucleotide polymorphism (SNP) detection by performing genome selection (GS) assistance GWAS. The results of the two GWAS analyses identified 55 and 40 SNPs that were respectively associated with plant height (PH) and ground diameter (GD), and 92 and 69 candidate genes, including 30 overlapping genes. The prediction accuracy of the GS model (rrBLUP) for phenotype exceeds 0.9. Transcriptome analysis of 13 genotypes under two N levels showed that genes related to carbon and N metabolism, amino acid metabolism, energy metabolism, and signal transduction were differentially expressed in the xylem of P. cathayana under N treatment. Furthermore, we observed strong regional patterns in gene expression levels of P. cathayana, with significant differences between different regions. Among them, P. cathayana in Longquan region exhibited the highest response to N. Finally, through weighted gene co-expression network analysis (WGCNA), we identified a module closely related to the N metabolic process and eight hub genes.ConclusionsIntegrating the GWAS, RNA-seq and WGCNA data, we ultimately identified four key regulatory genes (PtrNAC123, PtrNAC025, Potri.002G233100, and Potri.006G236200) involved in the wood formation process, and they may affect P. cathayana growth and wood formation by regulating nitrogen metabolism. This study will provide strong evidence for N regulation mechanisms, and reliable genetic resources for growth and NUE genetic improvement in poplar.

Comprehensive analysis of clinical indications and viral strain variants among patients infected with SARS-CoV-2 in Inner Mongolia, China

Since the first appearance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019, the virus is still evolving and mutating until now. In this study, we collected 6 throat swabs from patients who diagnosed with COVID-19 in Inner Mongolia, China, to understand the entry of multiple SARS-CoV-2 variants into Inner Mongolia and analyze the relationships between variants and clinical features observed in infected patients. In addition, we performed a combined analysis of clinical parameters associated with SARS-CoV-2 variants of interest, pedigree analysis, and detection of single-nucleotide polymorphisms. Our results showed that the clinical symptoms were generally mild although some patients demonstrated some degree of liver function abnormalities, and the SARS-CoV-2 strain was related to the Delta variant (B.1.617.2), AY.122 lineage. The epidemiological investigations and clinical manifestations confirmed that the variant exhibits strong transmission, a high viral load, and moderate clinical symptoms. SARS-CoV-2 has undergone extensive mutations in various hosts and countries. Timely monitoring of virus mutation can help to monitor the spread of infection and characterize the diversity of genomic variants, thus limiting future waves of SARS-CoV-2 infection.

Association of the rs2111234, rs3135499, rs8057341 polymorphisms in the NOD2 gene with leprosy: A case-control study in the Norte de Santander, Colombia population.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. The development of leprosy involves several factors, including the causative agent, the individual host's immune response, environmental factors, and the genetic background of the host. Specifically, the host's innate immune response, encoded by genes, determines their susceptibility to developing leprosy post-infection. Polymorphic variants in the nucleotide-binding oligomerization domain 2 (NOD2) gene are associated with leprosy among populations in a variety of endemic areas around the world. Colombia, a country located in the tropical zone, has several leprosy-endemic regions, including Norte de Santander. The aim of this study was to analyze the rs7194886, rs2111234, rs3135499, and rs8057341 single nucleotide polymorphisms (SNPs) in the NOD2 gene using a case-control study to determine whether they confer greater or lesser susceptibility to the development of leprosy. The TaqMan qPCR amplification system was used for SNPs detection. An association between the A-rs8057341 SNP (p = 0,006286) and resistance to leprosy was found. However, the rs3135499 (p = 0,9063) and rs2111234 (p = 0.1492) were not found to be associated with leprosy susceptibility. In addition, the rs7194886 SNP was not found to be in Hardy-Weinberg equilibrium (HWE) in the study population. The GAG haplotype, consisting of SNPs rs2111234-G, rs3135499-A, and rs8057341G, acts as a susceptibility factor for the development of leprosy in women. SNPs rs3135499 and rs8057341 are functionally related to decreased NOD2 expression according to an in-silico analysis. The SNPs rs8057341-A was related with resistance to leprosy and the haplotype rs2111234-G, rs3135499-A and rs8057341-G SNPs was related with susceptibility in the Norte de Santander Colombia, studied population.

Single-Nucleotide Polymorphisms, c.415C > T (Arg139Cys) and c.416G > A (Arg139His), in the NUDT15 Gene Are Associated with Thiopurine-Induced Leukopenia.

While nucleoside diphosphate-linked moiety X-type motif 15 (NUDT15) gene polymorphism Arg139Cys (rs116855232) is known to be a risk factor for thiopurine-induced severe leukopenia, association with the NUDT15 gene polymorphism Arg139His (rs147390019) has not yet been clarified. In addition, the accuracy of TaqMan PCR to assess these two polymorphisms has not been investigated. In this study, we evaluated TaqMan PCR for detection of the NUDT15 single-nucleotide polymorphisms (SNPs) and examined the clinical impact of Arg139His on thiopurine-induced leukopenia. First, we demonstrated that a TaqMan PCR assay successfully detected the Arg139His polymorphism of NUDT15 in clinical samples. Next, the NUDT15 gene polymorphisms (Arg139Cys and Arg139His) were separately analyzed by TaqMan Real-Time PCR in 189 patients from August 2018 to July 2019. The incidences of leukopenia within 2 years were 16.2, 57.9, and 100% for arginine (Arg)/Arg, Arg/cysteine (Cys), and Arg/histidine (His), respectively. The leukopenia was significantly increased in Arg/Cys and Arg/His compared with Arg/Arg. This retrospective clinical study indicated that, in addition to Arg139Cys, Arg139His may be clinically associated with a high risk of leukopenia. Pharmacogenomics will help in selecting drugs and determining the individualized dosage of thiopurine drugs.

Detection of single nucleotide polymorphism in HTR4 and its relationship with growth traits in sheep

In this study, a single nucleotide polymorphism of HTR4 (hydroxytryptamine receptor 4) was detected using DNA sequencing and KASPar (Kompetitive Allele-Specific PCR) technique with the aim of analyzing its effect on growth traits in 1102 sheep. A synonymous mutation g.101220 C > T located on the fifth intron of the ovis HTR4 gene was detected, and association analysis showed that this mutation was significantly associated with growth traits in sheep (p ＜.05), with TT genotypes having significantly lower body weight, height, length and chest circumference than TC and CC genotypes. It showed that the polymorphism of this locus was significantly associated with growth traits in sheep. In addition, qRT-PCR results showed that HTR4 was expressed in different tissues of sheep. It is highly expressed in the liver, spleen and duodenum. As important metabolic, immune and digestive absorption organs in animals, the above tissues can regulate the excitability of intestinal smooth muscle by participating in the body metabolism and nutrient metabolism of sheep, so that sheep can show better growth characteristics. In conclusion, the polymorphic locus identified in HTR4 gene can be used as candidate molecular marker in sheep breeding.

Detecting Melanocortin 1 Receptor Gene's SNPs by CRISPR/enAsCas12a.

Beyond its powerful genome-editing capabilities, the CRISPR/Cas system has opened up a new era of molecular diagnostics due to its highly specific base recognition and trans-cleavage activity. However, most CRISPR/Cas detection systems are mainly used to detect nucleic acids of bacteria or viruses, while the application of single nucleotide polymorphism (SNP) detection is limited. The MC1R SNPs were investigated by CRISPR/enAsCas12a and are not limited to the protospacer adjacent motif (PAM) sequence in vitro. Specifically, we optimized the reaction conditions, which proved that the enAsCas12a has a preference for divalent magnesium ion (Mg2+) and can effectively distinguish the genes with a single base difference in the presence of Mg2+, and the Melanocortin l receptor (MC1R) gene with three kinds of SNP sites (T305C, T363C, and G727A) was quantitatively detected. Since the enAsCas12a is not limited by PAM sequence in vitro, the method shown here can extend this extraordinary CRISPR/enAsCas12a detection system to other SNP targets, thus providing a general SNP detection toolbox.

Polymorphisms and gene expression of metalloproteinases and their inhibitors associated with cerebral ischemic stroke in young patients with sickle cell anemia.

Sickle cell anemia (SCA) is a genetic disease with great clinical heterogeneity and few viable strategies for treatment; hydroxyurea (HU) is the only widely used drug. Thus, the study of single nucleotide polymorphisms (SNPs) and the gene expression of MMPs 1, 2, 9, 7 and TIMPs 1 and 2, which are involved in the regulation of extracellular matrix, inflammation, and neuropathies, may provide further insights into the pathophysiology of the disease and elucidate biomarkers and molecules as potential therapeutic targets for patients with SCA. We evaluated 251 young individuals with SCA from northeastern Brazil. The groups were divided according to vaso-occlusive crisis (VOC) and cerebrovascular disease (CVD), compared to control individuals. SNP detection and gene expression assays were performed by real-time PCR, TaqMan system®. Both the expression levels of MMP1 gene, and the SNP MMP1-1607 1G/2G were associated with the risk of cerebral ischemic stroke (IS), and the expression of MMP1 was also associated with a higher frequency of VOC/year. Expression levels of MMP7, TIMP1, and TIMP2 were increased in patients conditioned to IS. The SNP 372T>C (rs4898) TIMP1 T alleles were more frequent in patients with > 5 VOC events/year. The SNP rs17576 of MMP9 showed differences in gene expression levels; it was increased in the genotypes AG, and AG+GG. The findings of this study, the SNPs, and expression provide initial support for understanding the role of MMPs-TIMPs in the pathophysiology of SCA in young patients.

SARS-CoV-2 genotyping and sequencing following a simple and economical RNA extraction and storage protocol.

Since the beginning of the SARS-CoV-2 pandemic, supply chain shortages have caused major disruptions in sourcing the materials needed for laboratory-based molecular assays. With increasing demand for molecular testing, these disruptions have limited testing capacity and hindered efforts to mitigate spread of the virus and new variants. Here we evaluate an economical and reliable protocol for the extraction and short-term ambient temperature storage of SARS-CoV-2 RNA. Additional objectives of the study were to evaluate RNA from this protocol for 1) detection of single nucleotide polymorphisms (SNPs) in the spike gene and 2) whole genome sequencing of SARS-CoV-2. The RNAES protocol was evaluated with residual nasopharyngeal (NP) samples collected from Emory Healthcare and Emory Student Health services. All RNAES extractions were performed in duplicate and once with a commercial extraction robot for comparison. Following extraction, eluates were immediately tested by rRT-PCR. SARS-CoV-2 RNA was successfully detected in 56/60 (93.3%) RNAES replicates, and Ct values corresponded with comparator results. Upon testing in spike SNP assays, three genotypes were identified, and all variant calls were consistent with those previously obtained after commercial extraction. Additionally, the SARS-RNAES protocol yield eluate pure enough for downstream whole genome sequencing, and results were consistent with SARS-CoV-2 whole genome sequencing of eluates matched for Ct value. With reproducible results across a range of virus concentrations, the SARS-RNAES protocol could help increase SARS-CoV-2 diagnostic testing and monitoring for emerging variants in resource-constrained communities.

Com probe implemented STexS II greatly enhances specificity in SARS-CoV-2 variant detection

The initial introduction of utilizing double helix structural oligonucleotides known as SNP typing with excellent specificity (STexS) in a standard PCR greatly improved the detection of single nucleotide polymorphisms (SNP) by enhancing amplification rates of primer-matching strands and interrupting mismatched strands by constant instability of kinetics regarding alignment attaching and detaching. The model was beneficial overall in detecting SNP variants consisting of large amounts of wildtype strands such as EGFR mutation genotyping for early detection of non-small cell lung cancer. While the STexS PCR is advantageous in detecting SNPs and biomarkers, limitations were yet observed. Despite the ability to detect variants 10 times more effective than a typical amplification-refractory mutation system PCR, it could only perform optimally in DNA concentrations around 101 ~ 105. To further enhance STexS specificity to perform detecting viral-RNA variants such as the infamous SARS-CoV-2, a novel improvement of the regular TaqMan Probe using Com-probes to inhibit high copy wild targets and amplify low copy mutant targets. By introducing the novel STexS II, omicron variants of SARS-CoV-2 were able to be successfully detected in high concentrations of normal genes.

A protein enzyme-free strategy for fluorescence detection of single nucleotide polymorphisms using asymmetric MNAzymes

To establish protein enzyme-free and simple approach for sensitive detection of single nucleotide polymorphisms (SNPs), the nucleic acid amplification reactions were developed to reduce the dependence on protein enzymes (polymerase, endonuclease, ligase). These methods, while enabling highly amplified analysis for the short sequences, cannot be generalized to long genomic sequences. Herein, we develop a protein enzyme-free and general SNPs assay based on asymmetric MNAzyme probes. The multi-arm probe (MNAzyme-9M-13) with two asymmetric recognition arms, containing a short (9 nt) and a long (13 nt) arm, is designed to detect EGFR T790 M mutation (MT). Owing to the excellent selectivity of short recognition arm, MNAzyme-9M-13 probe can efficiently avoid interferences from wild-type target (WT) and various single-base mutations. Through a one-pot mixing, MNAzyme-9M-13 probe enables the sensitive detection of MT, without protein enzyme or multi-step operation. The calculated detection limit for MT is 0.59 nM and 0.83%. Moreover, this asymmetric MNAzyme strategy can be applied for SNPs detection in long genomic sequences as well as short microRNAs (miRNAs) only by changing the low-cost unlabeled recognition arms. Therefore, along with simple operation, low-cost, protein enzyme-free and strong versatility, our asymmetric MNAzyme strategy provides a novel solution for SNPs detection and genes analysis.