Whole-cell SELEX Research Articles

Development of a whole-cell SELEX process to select species-specific aptamers against Aspergillus niger

BackgroundSpores produced by the filamentous fungus Aspergillus niger are abundant in a variety of environments. The proliferation of this fungus in indoor environments has been associated to health risks and its conidia can cause allergic reaction and severe invasive disease in animals and humans. Therefore, the detection and monitoring of Aspergillus conidia is of utmost importance to prevent serious fungal infections and contaminations. Among others, aptamers could serve as biosensors for the specific detection of fungal spores.ResultsIn this study, DNA aptamers specific to conidia of A. niger were developed by optimizing a whole-cell SELEX approach. Three whole-cells SELEX experiments were performed in parallel with similar conditions. Quantification of recovered ssDNA and melting curve analyses were applied to monitor the ongoing SELEX process. Next-generation sequencing was performed on selected recovered ssDNA pools, allowing the identification of DNA aptamers which bind with high affinity to the target cells. The developed aptamers were shown to be species-specific, being able to bind to A. niger but not to A. tubingensis or to A. nidulans. The binding affinity of two aptamers (AN01-R9-006 and AN02-R9-185) was measured to be 58.97 nM and 138.71 nM, respectively, which is in the range of previously developed aptamers.ConclusionsThis study demonstrates that species-specific aptamers can be successfully developed via whole-cell SELEX to distinguish different Aspergillus species and opens up new opportunities in the field of diagnostics of fungal infections.

Development of novel DNA aptamers and colorimetric nanozyme aptasensor for targeting multi-drug-resistant, invasive Salmonella typhimurium strain SMC25

Invasive, biofilm-forming Non-typhoidal Salmonella (iNTS), propagating through the global food and water supply chain, presents a significant risk to food safety and public health. Developing a robust detection system is crucial for enabling point-of-care, affordable, and equipment-free identification of this pathogen throughout the supply chain. In this study, we screened a novel pool of ssDNA aptamers specific to a multidrug resistant iNTS strain SMC25, previously isolated from Indian poultry products in our earlier research. Through 13 rounds of whole-cell SELEX, we identified, characterized, and selected seven full-length aptamers (ST18, ST19, ST25, ST28, ST29, ST31, and ST32). Flow cytometric analysis reveals superior binding of ST25, ST28, ST29, and ST31. These aptamers were translated onto Nanozyme-based aptasensing system for efficient, cost-effective detection of SMC25. This system harnesses the aptamer-mediated, reversible peroxidase-like activity of gold nanoparticles (GNPs) to oxidize the TMB substrate into a one-electron oxidation state, resulting in a blue-colored Diamine charge transfer complex (DCTC). The catalytic process, coupled with GNP aggregation, induces a visible color change in the test mixture from ruby-red to blue. Post-SELEX truncations identified the optimal aptamer sequence (T_ST31), which selectively detected SMC25 in water with a limit of detection (LOD) of ∼10⁴ CFU/mL. Lower concentrations (10 CFU/mL) of SMC25 could be detected after non-selective enrichment within 120 min. This research introduces a novel pool of iNTS-specific aptamers along with a cost-effective (0.25 USD per sample) solution for colorimetric detection by the naked eye.

SELEX against whole-cell bacteria resulted in lipopolysaccharide binding aptamers

Nucleic acid aptamers are target-specific oligonucleotides selected from combinatorial libraries through an iterative in vitro screening process known as Systemic Evolution of Ligands by Exponential Enrichment (SELEX). In this report, the selection of bacteria differentiating ssDNA aptamer candidates from a combinatorial library through the whole-cell SELEX method was performed. The enriched SELEX pool was sequenced using Illumina Next-Generation Sequencing (NGS) technology and analyzed for the most abundant sequences using CLC Genomics Workbench. The sequencing data resulted in several oligonucleotide families from which three individual sequences were chosen per SELEX based on the copy numbers. The binding performance of the selected aptamers was assessed by flow cytometry and fluorescence spectroscopy, and the binding constants were estimated using binding saturation curves. Varying results were obtained from two independent SELEX procedures where the SELEX against the model gram-negative bacterium Escherichia coli provided more selective sequences while the SELEX library used against gram-positive bacterium Listeria monocytogenes did not evolve as expected. The sequences that emerged from E. coli SELEX were shown to bind Lipopolysaccharide residues (LPS) and inhibit LPS-induced macrophage polarization. Thus, it can be said that, performed whole-cell SELEX could be resulted as the selection of aptamers which can bind LPS and inhibit LPS induced inflammation response and thus can be candidates for the inhibition of bacterial infections. In future studies, the selected aptamer sequences could be structurally and chemically modified and exploited as potential diagnostic tools and therapeutic agents as LPS antagonists.

DNA aptamers specific for Legionella pneumophila: systematic evolution of ligands by exponential enrichment in whole bacterial cells.

Legionella pneumophila is the major causative agent of Legionnaires' disease and Pontiac fever, which pose major public health problems. Rapid detection of L. pneumophila is important for global control of these diseases. Aptamers, short oligonucleotides that bind to targets with high affinity and specificity, have great potential for use in pathogenic bacterium detection, diagnostics, and therapy. Here, we used a whole-cell SELEX (systematic evolution of ligands by exponential enrichment) method to isolate and characterize single-stranded DNA (ssDNA) aptamers against L. pneumophila. A total of 60 ssDNA sequences were identified after 17 rounds of selection. Other bacterial species (Escherichia coli, Bacillus subtilis, Pseudomonas syringae, Staphylococcus aureus, Legionella quateirensis, and Legionella adelaidensis) were used for counterselection to enhance the specificity of ssDNA aptamers against L. pneumophila. Four ssDNA aptamers showed strong affinity and high selectivity for L. pneumophila, with Kd values in the nanomolar range. Bioinformatic analysis of the most specific aptamers revealed predicted conserved secondary structures that might bind to L. pneumophila cell walls. In addition, the binding of these four fluorescently labeled aptamers to the surface of L. pneumophila was observed directly by fluorescence microscopy. These aptamers identified in this study could be used in the future to develop medical diagnostic tools and public environmental detection assays for L. pneumophila.

Research progress of whole-cell-SELEX selection and the application of cell-targeting aptamer.

Aptamers refer to the artificially synthesized nucleic acid sequences (DNA/RNA) that can bind to a wide range of targets with high affinity and specificity, which are generally generated from systematic evolution of ligands by exponential enrichment (SELEX). As a novel method of aptamers screening, whole-cell-SELEX (WC-SELEX) has gained more and more attention in many fields such as biomedicine, analytical chemistry, and molecular diagnostics due to its ability to screen multiple potential aptamers without knowing the detailed structural information of target molecules. In recent years, with the deepening of research on application of aptamers, the traditional WC-SELEX cannot meet the practical application because of long experimental period, complicated operation process and low specificity, etc. Therefore, the development of more efficient methods for screening aptamer is always on the road. This paper summarizes the current research status of WC-SELEX for bacteria, parasites and animal cells, and reviews the latest advances of WC-SELEX techniques that are dependent on novel instruments, materials and microelectronics, including fluorescence-activated cell sorting-assisted SELEX, three-dimensional assisted WC-SELEX, and microfluidic chip system-assisted WC-SELEX. In addition, the application of aptamers targeting cells was discussed. Taken together, this review is aimed at providing a reference for WC-SELEX selection and application of aptamer targeting cells.

Highly adaptable and sensitive FRET-based aptamer assay for the detection of Salmonella paratyphi A.

Here we demonstrate a facile and versatile fluorescence resonance energy transfer (FRET) based aptasensor for rapid detection of Salmonella paratyphi A. The assay shows a detection limit up to 10cfu·mL-1 with no cross-reactivity with other bacterial species. Less than 8% of inter-assay coefficient variance and recovery rate between 85 and 102% attests the assay reliability. The advantages of FRET-based aptamer assay over the conventional immunoassay formats such as ELISA are the specificity, speed, reliability, and simplicity of the assay. The ssDNA aptamers specific towards pathogenic Salmonella paratyphi A were generated via whole-cell SELEX. The aptamer was conjugated onto quantum dot (QD) that served as the molecular beacon and graphene oxide (GO) was used as a fluorescence quencher. Thus the proposed method enables detection of target pathogen using FRET-based assay. Further interaction of aptamer with pathogen protein DNA gyrase was explored using classical molecular dynamics simulation.

Novel Aptamer-Functionalized Nanoparticles Enhances Bone Defect Repair By Improving Stem Cell Recruitment.

BackgroundThe restoration and repair method in the clinic of delayed fracture healing and non-union after comminuted fractures are urgently needed to improve the prognosis of patients. The recruitment of endogenous stem cells has been considered a promising approach in bone defect repair.ProposeThe aim of this study was to generate a de novel MSCs aptamer and developed the first, feasible, economical, bio-compatible, and functional MSCs aptamer-directed nanoparticles without complex manufacture to recruit mesenchymal stem cells (MSCs) for bone defect regeneration.MethodsWhole-cell SELEX was used to generate a de novel MSCs aptamer. Flow cytometry was applied to assess the binding specificities, affinities and sorting abilities of the aptamers. Nano-Aptamer Ball (NAB) was constructed by NHS/EDC reaction. The diameter and zeta of NAB were assessed by dynamic light scattering. CCK8 assay was utilized to evaluate whether NAB could cause non-specific cytotoxicity and induce cell proliferation. To evaluate the bone repair capacity of NAB, histomorphological staining, alizarin red and micro X-ray were used to observe the repair degree of defect in vivo. ELISA was used to detect osteopontin (OPN), osteocalcin (BGP) by, and alkaline phosphatase (ALP) in peripheral blood.ResultsMSCs aptamer termed as HM69 could bind with MSCs with high specificity and Kd of 9.67 nM, while has minimal cross-reactivities to other negative cells. HM69 could capture MSCs with a purity of >89%. In vitro, NAB could bind and capture MSCs effectively, whereas did not cause obvious cytotoxicity. In vivo, serum OPN, BGP, and ALP levels in the NAB group of rats were increased at both 2 and 4 weeks, indicating the repair and osteogenesis generation. The healing of bone defects in the NAB group was significantly better than control groups, the defects became blurred, and local trabecular bone growth could be observed in X-ray. The organized hematoma and cell growth in the bone marrow of the NAB group were more vigorous in bone sections staining.ConclusionThese suggested that HM69 and HM69-functionalized nanoparticles NAB exhibited the ability to recruit MSCs both in vitro and in vivo and achieved a better outcome of bone defect repair in a rat model. The findings demonstrate a promising strategy of using aptamer-functionalized bio-nanoparticles for the restoration of bone defects via aptamer-introduced homing of MSCs.

Novel Aptamer-Functionalized Nanoparticles Enhances Bone Defects Repair by Improving Stem Cell Recruitment

The restoration and repair method in clinic of delayed fracture healing and non-union after comminuted fractures are urgently needed to improve the prognosis of patients. Although recruitment of endogenous stem cells has been considered a promising approach in bone defect repair, the current methods are mainly dependent on bio-scaffolds which own disadvantages. Thus, a theranostic platform to overcome these inadequacies is needed indeed. Methods: In this study, using the whole-cell SELEX technique and chemical polymerization, we generated a de novel MSCs aptamer and developed the first, feasible, economical, bio-compatible, and functional MSCs aptamer-directed nanoparticles (Nano-Aptamer Ball, NAB) without complex manufacture to recruit mesenchymal stem cells (MSCs) for bone defect regeneration. Results: In vitro, this MSCs aptamer (termed as HM69) can bind with MSCs with high specificity and Kd of 9.67 nM, while has minimal cross reactivities to other negative cells. Further, aptamer HM69 could capture MSCs with a purity of >89%. In vivo, NAB enhanced MSCs homing in comparison with random DNA or saline, and exhibited superior cartilage restoration when compared with the control group. Principal conclusions: These suggested that HM69 and HM69-functionalized nanoparticles NAB exhibited the ability to recruit MSCs both in vitro and in vivo and achieved a better outcome of bone defect repair in a rat model. The findings demonstrate a promising strategy of using aptamer-functionalized bio-nanoparticles for restoration of bone defects via aptamer-introduced homing of MSCs. Funding Statement: This work was funded by Shaanxi key research and development program (2019SF-207& 2017SF-280); Xi’an Science and Technology program SF1510(4). Declaration of Interests: The authors declare no conflicts of interests. Ethical Approval Statement: Missing.

Aptamer-Based Pathogen Monitoring for Salmonella enterica ser. Typhimurium.

Salmonella enterica ser. Typhimurium is a foodborne pathogen that causes salmonellosis. Symptoms of salmonellosis include fever, diarrhea, and gastroenteritis. Conventional culture methods for detecting foodborne bacterial pathogens require long incubation time, expensive immunoassay methods, and sample enrichment steps. The objective of this study was to develop an aptamer-based sandwich assay to detect S. enterica ser. Typhimurium. First, S. enterica ser. Typhimurium specific binding aptamers (S11 and S24) were obtained by whole-cell SELEX for high sensitivity and specificity detection using live S. enterica ser. Typhimurium. S11 and S24 aptamers were able to capture S. enterica ser. Typhimurium selectively and distinguish it from other species of Salmonella (S. enterica ser. Typhimurium, S. enterica ser. Choleraesuis, S. enterica ser. Dublin, and S. enterica ser. Enteritidis) and food-borne bacterial pathogens (Escherichia coli K12, Listeria monocytogenes, Shigella sonnei and Staphylococcus aureus) with KD values of 4.41×10-12 M and 3.75×10-11 M, respectively. S. enterica ser. Typhimurium. aptamer-based sandwich assay exhibited a linear response for sensing S. enterica ser. Typhimurium. cells at concentration ranging from 2×101 to 2×105 CFU/mL. This aptamer-based sandwich assay can be used for on-site detection of S. enterica ser. Typhimurium.

Sperm cell purification from mock forensic swabs using SOMAmer™ affinity reagents

We have demonstrated a proof of concept with affinity-based purification of sperm cells from mock forensic samples using SOMAmer™ reagents, DNA-based affinity reagents developed by SomaLogic, Inc. SOMAmer reagents were selected in vitro using whole-cell SELEX to bind specifically with intact, detergent-treated sperm cells. Successful separation of sperm from epithelial cells and their debris was demonstrated using buccal swabs with added semen. Primarily male DNA profiles were generated from sperm cells eluted from the types of cotton swabs typically used for rape kit evidence collection. The quality of sperm DNA isolated from samples purified using SOMAmers is comparable to existing commercially available differential extraction-based methods at higher sperm concentrations. This purification method is simple, offers relatively rapid (<2hr) sperm purification, and can potentially be automated using robotic workstations. This work serves as proof of concept that demonstrates the first use of SOMAmer reagents as affinity ligands to bind sperm cells. With further development, this technique can potentially be used for high-throughput sexual assault forensic casework.

DNA aptamers from whole-cell SELEX as new diagnostic agents against glioblastoma multiforme cells.

Glioma is a cancer derived from transformed glial cells, which are often invasive and display a heterogeneous cell population. Currently, no trustworthy biomarkers for the detection and risk stratification of glioma have been discovered. The objective of the present research was to select DNA aptamers to facilitate early diagnosis and effective therapy of glioma. Using cell-SELEX, three aptamers (WYZ-37, WYZ-41, WYZ-50), which can specifically recognize the molecular differences between target cells T98G and negative cells SVGp12, were identified. The best binding sequences WYZ-41 and WYZ-50 were optimized in length, resulting in aptamer sequences WYZ-41a and WYZ-50a. The Kd values of the aptamers WYZ-41a and WYZ-50a against the target cell line were found to be 1.0 ± 0.2 nM and 2.8 ± 0.6 nM, respectively, which are better than the Kds for full-length aptamers WYZ-41 and WYZ-50. Flow cytometry analysis results show that the aptamers WYZ-41a and WYZ-50a do not influence each other in mutual binding, and that they effectively detect the target even in complex mixtures, such as undiluted fetal bovine serum (FBS) and cerebral spinal fluid (CSF), indicating that aptamers WYZ-41a and WYZ-50a have excellent potential as aptamer pairs to improve the accuracy of glioma diagnosis.

In vitro selection of cell-internalizing 2′-modified RNA aptamers against Pseudomonas aeruginosa

Infectious diseases caused by bacterial or viral agents represent the major cause of human pathogenesis and mortality worldwide. A development of novel antibacterial therapeutics and diagnostic tools is a very acute task. The use of DNA and RNA aptamers targeted to certain bacteria could be a promising solution to this problem. Here, we propose a new protocol of selection of 2′-fluoro RNA aptamers capable to internalize into bacterial cells. Using whole-cell SELEX against Pseudomonas aeruginosa, enriched 2′-fluoro RNA library was obtained, and its sequencing and data analysis were fulfilled. It was found that the central region of predominating aptamer sequence is identical to the fragment of P. aeruginosa rRNA. A possibility of internalizing of this aptamer into bacterial cells is shown. It is hypothesized that aptamers could be internalized more effectively as heterodimeric complexes.

197 - Whole-Cell SELEX method modification for generation of nucleotide-modified RNA-aptamers to the cell surface: Application in Burkitt lymphoma versus non-malignant lymphoblastoid cells model

197 - Whole-Cell SELEX method modification for generation of nucleotide-modified RNA-aptamers to the cell surface: Application in Burkitt lymphoma versus non-malignant lymphoblastoid cells model

Aptamer-Based Technologies in Foodborne Pathogen Detection.

Aptamers are single stranded DNA or RNA ligands, which can be selected by a method called systematic evolution of ligands by exponential enrichment (SELEX); and they can specifically recognize and bind to their targets. These unique characteristics of aptamers offer great potentials in applications such as pathogen detection and biomolecular screening. Pathogen detection is the critical means in detecting and identifying the problems related to public health and food safety; and only the rapid, sensitive and efficient detection technologies can enable the users to make the accurate assessments on the risks of infections (humans and animals) or contaminations (foods and other commodities) caused by various pathogens. This article reviews the development in the field of the aptamer-based approaches for pathogen detection, including whole-cell SELEX and Genomic SELEX. Nowadays, a variety of aptamer-based biosensors have been developed for pathogen detection. Thus, in this review, we also cover the development in aptamer-based biosensors including optical biosensors for multiple pathogen detection by multiple-labeling or label-free models such as fluorescence detection and surface plasmon resonance, electrochemical biosensors and lateral chromatography test strips, and their applications in pathogen detection and biomolecular screening. While notable progress has been made in the field in the last decade, challenges or drawbacks in their applications such as pathogen detection and biomolecular screening remain to be overcome.

Improved Aptamers for the Diagnosis and Potential Treatment of HER2-Positive Cancer.

Aptamers provide a potential source of alternative targeting molecules for existing antibody diagnostics and therapeutics. In this work, we selected novel DNA aptamers targeting the HER2 receptor by an adherent whole-cell SELEX approach. Individual aptamers were identified by next generation sequencing and bioinformatics analysis. Two aptamers, HeA2_1 and HeA2_3, were shown to bind the HER2 protein with affinities in the nanomolar range. In addition, both aptamers were able to bind with high specificity to HER2-overexpressing cells and HER2-positive tumor tissue samples. Furthermore, we demonstrated that aptamer HeA2_3 is being internalized into cancer cells and has an inhibitory effect on cancer cell growth and viability. In the end, we selected novel DNA aptamers with great potential for the diagnosis and possible treatment of HER2-positive cancer.

The application of capillary electrophoresis for assisting whole-cell aptamers selection by characterizing complete ssDNA distribution

Whole-cell SELEX faces more difficulties than SELEX against purified molecules target. In this work, we demonstrate the application of capillary electrophoresis for assisting whole-cell aptamers selection by characterizing complete ssDNA distribution. We chose three cancer cell lines U251, Hela and PC3 as target, FAM labeled Sgc8c (a 41mer aptamer) and FAM labeled 41mer random ssDNA library as ssDNA model. CE conditions of running buffer and capillary length and inner diameter as well as UV and LIF detection were optimized. The distribution percentage of Sgc8c and ssDNA library against U251, Hela and PC3 was demonstrated, the relative peak area of their complex is 8.94%, 1.05% and 0.44% for Sgc8c and 9.03%, 1.04% and 0.12% for ssDNA library respectively. Under the chosen experimental conditions, binding ability comparison of three cell lines was U251>Hela>PC3, which was validated by laser confocol microscope. For each cell, distribution percentage of ssDNA library was compared with that of Sgc8c. Finally, whole-cell complex of U251–Sgc8c was confirmed by increase incubation time and fraction CE analysis.

DNA aptamers for the detection of Haemophilus influenzae type b by cell SELEX.

Haemophilus influenzae type b (Hib) causes acute bacterial meningitis (ABM) in children, with a mortality rate of about 3-6% of the affected patients. ABM can lead to death during a period of hours to several days and, hence, rapid and early detection of the infection is crucial. Aptamers, the short single-stranded DNA or RNA with high affinity to target molecules, are selected by a high-flux screening technique known as in vitro screening and systematic evolution of ligands by exponential enrichment technology (SELEX). In this study, whole-cell SELEX was applied for the selection of target-specific aptamers with high affinity to Hib. ssDNA aptamers prepared by lambda exonuclease were incubated with the target cells (Hib). The aptameric binding rate to Hib was characterized for binding affinity after seven SELEX rounds by flow cytometry. The aptamers with higher binding affinity were cloned. Four of 68 aptamer clones were selected for sequencing. The dissociation constant (Kd) of the high-affinity aptamer clones 45 and 63 were 47.10 and 28.46pM, respectively. These aptamers did not bind to other bacterial species, including the seven meningitis-causing bacteria. They showed distinct affinity to various H. influenzae strains only. These aptamers showed the highest affinity to Hib and the lowest affinity to H. influenzae type c and to other meningitis-causing bacteria. Clone 63 could detect Hib in patients' cerebrospinal fluid (CSF) samples at 60colony-forming units (CFU)/mL. The results indicate applicability of the aptamers for rapid and early detection of infections brought about by Hib.

Comparison of whole-cell SELEX methods for the identification of Staphylococcus aureus-specific DNA aptamers.

Whole-cell Systemic Evolution of Ligands by Exponential enrichment (SELEX) is the process by which aptamers specific to target cells are developed. Aptamers selected by whole-cell SELEX have high affinity and specificity for bacterial surface molecules and live bacterial targets. To identify DNA aptamers specific to Staphylococcus aureus, we applied our rapid whole-cell SELEX method to a single-stranded ssDNA library. To improve the specificity and selectivity of the aptamers, we designed, selected, and developed two categories of aptamers that were selected by two kinds of whole-cell SELEX, by mixing and combining FACS analysis and a counter-SELEX process. Using this approach, we have developed a biosensor system that employs a high affinity aptamer for detection of target bacteria. FAM-labeled aptamer sequences with high binding to S. aureus, as determined by fluorescence spectroscopic analysis, were identified, and aptamer A14, selected by the basic whole-cell SELEX using a once-off FACS analysis, and which had a high binding affinity and specificity, was chosen. The binding assay was evaluated using FACS analysis. Our study demonstrated the development of a set of whole-cell SELEX derived aptamers specific to S. aureus; this approach can be used in the identification of other bacteria.

Selection and characterization of DNA aptamers specific for Listeria species

Single-stranded (ss) DNA aptamers with binding affinity to Listeria spp. were selected using a whole-cell SELEX (Systematic Evolution of Ligands by EXponential enrichment) method. Listeria monocytogenes cells were grown at 37°C and harvested at mid-log phase or early stationary phase to serve as the targets in SELEX. A total of 10 unique aptamer sequences were identified, six associated with log phase cells and four with stationary phase cells. Binding affinity of the aptamers was determined using flow cytometry and ranged from 10% to 44%. Four candidates having high binding affinity were further studied and found to show genus-specific binding affinity when screened against five different species within the Listeria genus. Using sequential binding assays combined with flow cytometry, it was determined that three of the aptamers (LM6-2, LM12-6, and LM12-13) bound to one apparent cell surface moiety, while a fourth aptamer (LM6-116) appeared to bind to a different cell surface region. This is the first study in which SELEX targeted bacterial cells at different growth phases. When used together, aptamers that bind to different cell surface moieties could increase the analytical sensitivity of future capture and detection assays.

In vitro Selection of DNA Aptamers and Fluorescence-Based Recognition for Rapid Detection Listeria monocytogenes

Aptamers are specific nucleic acid sequences that can bind to a wide range of nucleic acid and non-nucleic acid targets with high affinity and specificity. Nucleic acid aptamers are selected in vitro from single stranded DNA or RNA ligands containing random sequences of up to a few hundred nucleotides. Systematic evolution of ligands by exponential enrichment (SELEX) was used to select and PCR amplify DNA sequences (aptamers) capable of binding to and detecting Listeria monocytogenes, one of the major food-borne pathogens. A simplified affinity separation approach was employed, in which L. monocytogenes in exponential (log) phase of growth was used as the separation target. A fluorescently-labeled aptamer assay scheme was devised for detecting L. monocytogenes. This report described a novel approach to the detection of L. monocytogenes using DNA aptamers. Aptamers were developed by nine rounds of SELEX. A high affinity aptamer was successfully selected from the initial random DNA pool, and its secondary structure was also investigated. One of aptamers named e01 with the highest affinity was further tested in aptamer-peroxidase and aptamer-fluorescence staining protocols. This study has proved the principle that the whole-cell SELEX could be a promising technique to design aptamer-based molecular probes for dectection of pathogenic microorganisms without tedious isolation and purification of complex markers or targets.