Browse
Submit Data
Databases
API
Help

Dataset Information

31 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Immuno Affinity SELEX for Simple, Rapid, and Cost-Effective Aptamer Enrichment and Identification against Aflatoxin B1.

ABSTRACT: Aflatoxins are naturally occurring mycotoxins that contaminate food and agro commodities, leading to acute and chronic health conditions in human and animals. In the present work, an attempt was made to generate high-affinity single stranded DNA aptamers that specifically bind to Aflatoxin B1 (AFB1) by a modified Systemic Evolution of Ligands by Exponential Enrichment (SELEX) procedure with the aid of Immunoaffinity columns. Ten rounds of SELEX and alternating three counter SELEX rounds with a cocktail of related and other mycotoxins were performed to enhance the specificity. Resultant 105 aptamers were clustered into 12 groups according to their primary sequence homology. Candidates with lowest Gibbs free energy (dG value) and unique stem loop structures were selected for further characterization. Aptamers, AFLA5, AFLA53, and AFLA71 exhibiting lower K_d values (50.45 ± 11.06, 48.29 ± 9.45, and 85.02 ± 25.74 nM) were chosen for development of ELONA and determination of purification ability of toxin. The detection limit (LOD) of AFLA5 and AFLA71 was 20 and 40 ng/ml, respectively. HPLC analysis implied that selected aptamers were able to recover and quantify 82.2 to 96.21% (LOQ - 53.74 ng) and 78.3 to 94.22% (LOQ - 66.75 ng) of AFB1 from spiked corn samples, respectively. These findings indicate, immunoaffinity based SELEX can pave an alternative approach to screen aptamers against mycotoxin detection and purification.

SUBMITTER: Setlem K

PROVIDER: S-EPMC5130984 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Aptamer against Aflatoxin B1 Obtained by SELEX and Applied in Detection.

Project description:Aflatoxins, especially aflatoxin B1 (AFB1), are the most prevalent mycotoxins in nature. They contaminate various crops and cause global food and feed safety concerns. Therefore, a simple, rapid, sensitive, and specific AFB1 detection tool is urgently needed. Aptamers generated by SELEX technology can specifically bind the desired targets with high affinity. The broad range of targets expands the scope of applications for aptamers. We used an AFB1-immobilized magnetic nanoparticle for SELEX to select AFB1-specific aptamers. One aptamer, fl-2CS1, revealed a dissociation constant (Kd = 2.5 μM) with AFB1 determined by isothermal titration calorimetry. Furthermore, no interaction was shown with other toxins (AFB2, AFG1, AFG2, OTA, and FB1). According to structural prediction and analysis, we identified a short version of the AFB1-specific aptamer, fl-2CS1/core, with a minimum length of 39-mer used in the AFB1-aptasensor system by real-time qPCR. The aptasensor showed a broad range of detection from 50 ppt to 50 ppb with an accuracy of 90% in the spiked peanut extract samples. With the application of the AFB1-aptasensor we have constructed, a wide range detection tool with high accuracy might be developed as a point-of-care testing tool in agriculture.

| S-EPMC9599246 | biostudies-literature

A Simple Structure-Switch Aptasensor Using Label-Free Aptamer for Fluorescence Detection of Aflatoxin B1.

Project description:Aflatoxin B1 (AFB1) is one of the mycotoxins produced by Aspergillus flavus and Aspergillus parasiticus, and it causes contamination in foods and great risk to human health. Simple sensitive detection of AFB1 is important and demanded for food safety and quality control. Aptamers can specifically bind to targets with high affinity, showing advantages in affinity assays and biosensors. We reported an aptamer structure-switch for fluorescent detection of aflatoxin B1 (AFB1), using a label-free aptamer, a fluorescein (FAM)-labeled complementary strand (FDNA), and a quencher (BHQ1)-labeled complementary strand (QDNA). When AFB1 is absent, these three strands assemble into a duplex DNA structure through DNA hybridization, making FAM close to BHQ1, and fluorescence quenching occurs. In the presence of AFB1, the aptamer binds with AFB1, instead of hybridizing with QDNA. Thus, FAM is apart from BHQ1, and fluorescence increases with the addition of AFB1. This assay allowed detection of AFB1 with a detection limit of 61 pM AFB1 and a dynamic concentration range of 61 pM to 4 μM. This aptamer-based method enabled detection of AFB1 in complex sample matrix (e.g., beer and corn flour samples).

| S-EPMC9268478 | biostudies-literature

Probing the limits of aptamer affinity with a microfluidic SELEX platform.

Project description:Nucleic acid-based aptamers offer many potential advantages relative to antibodies and other protein-based affinity reagents, including facile chemical synthesis, reversible folding, improved thermal stability and lower cost. However, their selection requires significant time and resources and selections often fail to yield molecules with affinities sufficient for molecular diagnostics or therapeutics. Toward a selection technique that can efficiently and reproducibly generate high performance aptamers, we have developed a microfluidic selection process (M-SELEX) that can be used to obtain high affinity aptamers against diverse protein targets. Here, we isolated DNA aptamers against three protein targets with different isoelectric points (pI) using a common protocol. After only three rounds of selection, we discovered novel aptamer sequences that bind to platelet derived growth factor B (PDGF-BB; pI?=?9.3) and thrombin (pI?=?8.3) with respective dissociation constants (K(d)) of 0.028 nM and 0.33 nM, which are both superior to previously reported aptamers against these targets. In parallel, we discovered a new aptamer that binds to apolipoprotein E3 (ApoE; pI?=?5.3) with a K(d) of 3.1 nM. Furthermore, we observe that the net protein charge may exert influence on the affinity of the selected aptamers. To further explore this relationship, we performed selections against PDGF-BB under different pH conditions using the same selection protocol, and report an inverse correlation between protein charge and aptamer K(d).

| S-EPMC3215713 | biostudies-literature

Recent Advances in the Aptamer-Based Electrochemical Biosensors for Detecting Aflatoxin B1 and Its Pertinent Metabolite Aflatoxin M1.

Project description:The notable toxicological impacts of aflatoxin B1 (AFB1) and its main metabolite, aflatoxin M1 (AFM1), on human being health make the evaluation of food quality highly significant. Due to the toxicity of those metabolites-even very low content in foodstuffs-it is crucial to design a sensitive and reliable procedure for their detection. Electrochemical aptamer-based biosensors are considered the most encouraging option, based on multi-placed analysis, rapid response, high sensitivity and specificity. The present review specifically emphasizes the potential utilization of the electrochemical aptasensors for determining the AFM1 and AFB1 with different electrodes.

| S-EPMC7309004 | biostudies-literature

Ribo-Pop: Simple, cost-effective, and widely applicable ribosomal RNA depletion

Project description:The measurement of RNA abundance derived from massively parallel sequencing experiments is an essential technique. Methods that reduce ribosomal RNA levels are usually required prior to sequencing library construction because ribosomal RNA typically comprises >90% of the total RNA molecules in a sample. For some experiments, ribosomal RNA depletion is favored over poly(A) selection because it offers a more inclusive representation of the transcriptome. However, methods to deplete ribosomal RNA are generally proprietary, complex, inefficient, applicable to only specific species, or compatible with only a narrow range of RNA input levels. Here, we describe Ribo-Pop (ribosomal RNA depletion for popular use), a simple workflow and antisense oligo design strategy that we demonstrate works over a wide input range and can be easily adapted to any organism with a sequenced genome. We provide a computational pipeline for probe selection, a streamlined 20-minute protocol, and ready-to-use oligo sequences for several organisms. We anticipate that our simple and generalizable “open source” design strategy would enable virtually any lab to pursue full transcriptome sequencing in their organism of interest with minimal time and resources.

2020-08-12 | GSE150332 | GEO

Effective Biodegradation of Aflatoxin B1 Using the Bacillus licheniformis (BL010) Strain.

Project description:Aflatoxin B1 (AFB1), a pollutant of agricultural products, has attracted considerable attention in recent years, due to its potential impact on health. In the present study, Bacillus licheniformis (BL010) was demonstrated to efficiently degrade AFB1, reducing over 89.1% of the toxin content within 120 h. A crude enzyme solution of BL010 exhibited the highest degradation level (97.3%) after three induction periods. However, uninduced BL010 bacteria was not capable of reducing AFB1. Furthermore, high performance liquid chromatography (HPLC) analysis showed that while a cell-free extract caused a significant decrease in AFB1 content (93.6%, p < 0.05), cell culture fluid treatment did not significantly degrade AFB1. The biotransformation products of AFB1 were detected and further identified by quadrupole time-of-flight liquid chromatography?mass spectrometry (LC-Q-TOF/MS); these corresponded to a molecular formula of C12H14O?. A sequence analysis of whole BL010 genes with a bioinformatics approach identified the secondary structures of two degrading enzymes (Chia010 and Lac010), providing an important basis for subsequent homology modeling and functional predictions.

| S-EPMC6315853 | biostudies-literature

Microfluidic paper device for rapid detection of aflatoxin B1 using an aptamer based colorimetric assay.

Project description:Contamination of milk by mycotoxins is a serious problem worldwide. Herein, we focused on the detection of aflatoxin B1 (AflB1) using a paper microfluidic device fabricated with specific aptamers as biorecognition elements. The fabrication process resulted in the generation of a leak proof microfluidic device where two zones were designed: control and test. Detection is achieved by color change when aflatoxin reacts with an aptamer followed by salt induced aggregation of gold nanoparticles. Specific aptamers for aflatoxin B1 were immobilized successfully onto the surface of gold nanoparticles. Biophysical characterization of the conjugated AuNPs-aptamer was done by UV-vis spectroscopy, DLS (dynamic light scattering), TEM (transmission electron microscopy). Under optimal conditions, the microfluidic device showed an excellent response for aflatoxin B1 detection in the range of 1 pM to 1 μM with a detection limit of up to 10 nM in spiked samples. Disadvantages associated with conventional techniques of ELISA were overcome by this one step detection technique with low operation cost, simple instrumentation, and user-friendly format with no interference due to chromatographic separation. The developed microfluidic paper-based analytical device (μPAD) can provide a tool for on-site detection of food toxins in less than a minute which is the main requirement for both qualitative and quantitative analysis in food safety and environmental monitoring.

| S-EPMC9050516 | biostudies-literature

Simultaneous Rapid Detection of Aflatoxin B1 and Ochratoxin A in Spices Using Lateral Flow Immuno-Chromatographic Assay.

Project description:Spices are susceptible to contamination by aflatoxin B1 (AFB1) and ochratoxin A (OTA), which are both mycotoxins with high toxicity and carcinogenicity. In this study, we aimed to develop an immuno-chromatographic strip test for the simultaneous quantification of AFB1 and OTA in spices by spraying the coupled antigens AFB1-ovalbumin (AFB1-OVA) and OTA-ovalbumin (OTA-OVA) on a nitrocellulose membrane. The test strip had high sensitivity, good specificity, and strong stability. The detection limits of these two mycotoxins in Chinese prickly ash, pepper, chili, cinnamon, and aniseed were 5 μg/kg. The false positivity rate was 2%, and the false negativity rate was 0%. The maximum coefficient of variation was 4.28% between batches and 5.72% within batches. The average recovery rates of AFB1 and OTA in spices were 81.2-113.7% and 82.2-118.6%, respectively, and the relative standard deviation (RSD) was <10%. The actual sample detection was consistent with high performance liquid chromatography analysis results. Therefore, the immuno-chromatographic test strips developed in this study can be used for the on-site simultaneous detection of AFB1 and OTA in spices. This method would allow the relevant regulatory agencies to strengthen supervision in an effort to reduce the possible human health hazards of such contaminated spices.

| S-EPMC8623159 | biostudies-literature

A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies

Project description:These experiments were designed to quantify depletion of rRNA sequencing reads from bacterial RNA-seq libraries and verify that mRNA sequencing reads were not altered. Specifically, we tested an rRNA depletion method using custom-designed biotinylated oligonucleotides and compared these results to undepleted (total RNA) libraries and libraries made with the previously-available Ribo-Zero kit (Illumina).

2019-12-28 | GSE142656 | GEO

Endonuclease enrichment TAPS for cost-effective genome-wide base-resolution DNA methylation detection

Project description:We described endonuclease enrichment TAPS (eeTAPS), which utilizes dihydrouracil (DHU)-sensitive endonuclease digestion after TAPS to enrich methylated CpG (mCpG). eeTAPS can accurately detect 87% of methylated CpGs in the mouse genome with a sequencing depth comparable to 4x whole genome sequencing. In comparison, reduced representation TAPS (rrTAPS) detected less than 4% of methylated CpGs with 2.5x sequencing depth. Our results demonstrate eeTAPS to be a new strategy for cost-effective genome-wide methylation analysis at single-CpG resolution that can fill the gap between whole-genome and reduced representation sequencing.

2021-04-01 | GSE148197 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data