Selection of DNA aptamers for capture and detection of Salmonella Typhimurium using a whole-cell SELEX approach in conjunction with cell sorting

Alternative ligands such as nucleic acid aptamers can be used for pathogen capture and detection and offer advantages over antibodies, including reduced cost, ease of production and modification, and improved stability. DNA aptamers demonstrating binding specificity to Salmonella enterica serovar Typhimurium were identified by whole-cell-systematic evolution of ligands by exponential enrichment (SELEX) beginning with a combinatorial library of biotin-labeled single stranded DNA molecules. Aptamer specificity was achieved using whole-cell counter-SELEX against select non-Salmonella genera. Aptamers binding to Salmonella were sorted, cloned, sequenced, and characterized for binding efficiency. Out of 18 candidate aptamers screened, aptamer S8-7 showed relatively high binding affinity with an apparent dissociation constant (K _d value) of 1.73?±?0.54 μM and was selected for further characterization. Binding exclusivity analysis of S8-7 showed low apparent cross-reactivity with other foodborne bacteria including Escherichia coli O157: H7 and Citrobacter braakii and moderate cross-reactivity with Bacillus cereus. Aptamer S8-7 was successfully used as a ligand for magnetic capture of serially diluted Salmonella Typhimurium cells, followed by downstream detection using qPCR. The lower limit of detection of the aptamer magnetic capture-qPCR assay was 10²–10³?CFU equivalents of Salmonella Typhimurium in a 290-μl sample volume. Mean capture efficiency ranged from 3.6 to 12.6 %. Unique aspects of the study included (a) the use of SELEX targeting whole cells; (b) the application of flow cytometry for aptamer pool selection, thereby favoring purification of ligands with both high binding affinity and targeting abundant cell surface moieties; and (c) the use of pre-labeled primers that circumvented the need for post-selection ligand labeling. Taken together, this study provides proof-of-concept that biotinylated aptamers selected by whole-cell SELEX can be used in a qPCR-based capture-detection platform for Salmonella Typhimurium.     

SELEX法筛选宫颈癌前病变核酸适配子

构建随机ssDNA文库,通过SELEX技术,以正常、炎性宫颈脱落细胞为反筛细胞,以上皮内低级别病变(CIN1)、上皮内高级别病变(CIN2、CIN3)和鳞状细胞癌脱落细胞为正筛细胞,经过12轮筛选特异性适配子高度富集得到宫颈癌前病变适配子库,经特异性、亲和力分析和细胞免疫荧光确立高特异性适配子CIN-Ap4可作为诊断宫颈癌前病变生物标志物,为宫颈癌前病变分子诊断奠定理论基础,提供新思路。利用Prime Premier 5.0设计构建了随机ssDNA文库并根据文库两端固定序列设计引物,对对称PCR和间接不对称PCR中的退火温度、循环数以及上、下游引物浓度比等条件进行优化,分析确定50μL反应体系中对称PCR的最佳反应条件为:最佳退火温度为49.5℃,最佳循环数为15个循环;间接不对称PCR的最佳反应条件为:50μL反应体系中上、下游引物浓度的最佳比例为80∶1,最佳循环数为35个循环。实验结果表明成功构建了寡核苷酸文库,在最适PCR条件下可获得理想的dsDNA和ssDNA,并具有良好的重复性,为顺利筛选适配子提供保证。     

Selection of aptamers against KMP-11 using colloidal gold during the SELEX process

SELEX procedure is a methodology in which single stranded oligonucleotides are selected from a wide variety of sequences based on their interaction with a target molecule. We have designed a novel SELEX methodology using colloidal gold to select high affinity single stranded DNA aptamers against Leishmania infantum KMP-11. Kinetoplastid membrane protein-11 (KMP-11) is a major component of the cell membrane of kinetoplastid parasites. Although its function is not known, the fact that KMP-11 is a cytoskeleton-associated protein suggests that it may be involved in mobility or in some other aspects of the flagellar structure. We have isolated a single stranded DNA aptamer population that binds specifically to L. infantum KMP-11. This population has been characterized in a series of in vitro experiments suggesting that it may be used as a powerful tool to further investigate the role of KMP-11 during Leishmania development and/or as a diagnostic tool in Leishmania infection.     

Selection of DNA aptamers using atomic force microscopy

Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an ‘AFM-SELEX’ cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules.     

High-throughput quantitative binding analysis of DNA aptamers using exonucleases

Aptamers are nucleic acid bioreceptors that have been used in various applications including medical diagnostics and as therapeutic agents. Identifying the most optimal aptamer for a particular application is very challenging. Here, we for the first time have developed a high-throughput method for accurately quantifying aptamer binding affinity, specificity, and cross-reactivity via the kinetics of aptamer digestion by exonucleases. We demonstrate the utility of this approach by isolating a set of new aptamers for fentanyl and its analogs, and then characterizing the binding properties of 655 aptamer–ligand pairs using our exonuclease digestion assay and validating the results with gold-standard methodologies. These data were used to select optimal aptamers for the development of new sensors that detect fentanyl and its analogs in different analytical contexts. Our approach dramatically accelerates the aptamer characterization process and streamlines sensor development, and if coupled with robotics, could enable high-throughput quantitative analysis of thousands of aptamer–ligand pairs.     

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.     

DNA identification and characterization of Campylobacter jejuni and Campylobacter coli isolated from caecal samples of chickens in Grenada

Aims: To speciate Campylobacter strains from the caeca of chickens in Grenada using PCR and to evaluate DNA‐based typing methods for the characterization of these isolates. Methods and Results: Isolates were speciated with two multiplex PCR assays and were typed with flaA‐RFLP, pulsed‐field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Results confirmed that Campylobacter coli strains were more predominant than Campylobacter jejuni strains. From 56 isolates, 18 were misidentified using biochemical tests. PFGE typing gave the highest discriminatory power among the methods used (Simpson’s index of diversity, D = 0·9061). However, the combination of flaA‐RFLP, PFGE and MLST results gave the highest discrimination for subtyping of these isolates (D = 0·9857). A band position tolerance of 4% in Bio Numerics was the most appropriate for the analysis of this database. MLST profiles were generally concordant with PFGE and/or flaA‐RFLP types. Several isolates exhibited new MLST sequence types (STs), and 43 of the 49 Camp. coli strains belonged to the ST‐828 clonal complex. Conclusions: Campylobacter coli was the most prevalent species isolated from broilers and layers in Grenada, and a combination of restriction and sequence methods was most appropriate for the typing of Camp. coli isolates. Campylobacter coli STs clustered with described poultry‐associated Camp. coli STs by phylogenetic analysis. Significance and Impact of the Study: Further studies to understand the predominance of Camp. coli within Campylobacter spp. from chickens in Grenada may help elucidate the epidemiology of these pathogens in chickens.     

Comparative genome analysis of Campylobacter jejuni using whole genome DNA microarrays

Whole genome DNA microarrays were constructed and used to investigate genomic diversity in 18 Campylobacter jejuni strains from diverse sources. New algorithms were developed that dynamically determine the boundary between the conserved and variable genes. Seven hypervariable plasticity regions (PR) were identified in the genome (PR1 to PR7) containing 136 genes (50%) of the variable gene pool. When comparisons were made with the sequenced strain NCTC11168, the number of absent or divergent genes ranged from 2.6% (40 genes) to 10.2% (163) and in total 16.3% (269) of the genes were variable. PR1 contains genes important in the utilisation of alternative electron acceptors for respiration and may confer a selective advantage to strains in restricted oxygen environments. PR2, 3 and 7 contain many outer membrane and periplasmic proteins and hypothetical proteins of unknown function that might be linked to phenotypic variation and adaptation to different ecological niches. PR4, 5 and 6 contain genes involved in the production and modification of antigenic surface structures.     

Physiological characterization of viable-but-nonculturable Campylobacter jejuni cells

Campylobacter jejuni is a pathogenic, microaerophilic, gram-negative, mesophilic bacterium. Three strains isolated from humans with enteric campylobacteriosis were able to survive at high population levels (10(7) cells ml-1) as viable-but-nonculturable (VBNC) forms in microcosm water. The VBNC forms of the three C. jejuni strains were enumerated and characterized by using 5-cyano-2,3-ditolyl tetrazolium chloride-4',6-diamino-2-phenylindole staining. Cellular volume, adenylate energy charge, internal pH, intracellular potassium concentration, and membrane potential values were determined in stationary-phase cell suspensions after 48 h of culture on Columbia agar and after 1 to 30 days of incubation in microcosm water and compared. A notable increase in cell volume was observed with the VBNC state; the average cell volumes were 1.73 microliter mg of protein-1 for the culturable form and 10.96 microliter mg of protein-1 after 30 days of incubation in microcosm water. Both the internal potassium content and the membrane potential were significantly lower in the VBNC state than in the culturable state. Culturable cells were able to maintain a difference of 0.6 to 0.9 pH unit between the internal and external pH values; with VBNC cells this difference decreased progressively with time of incubation in microcosm water. Measurements of the cellular adenylate nucleotide concentrations revealed that the cells had a low adenylate energy charge (0.66 to 0.26) after 1 day of incubation in microcosm water, and AMP was the only nucleotide detected in the three strains after 30 days of incubation in microcosm water.     

Selection of RNA aptamers against the M. tuberculosis EsxG protein using surface plasmon resonance-based SELEX

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis, remains one of the most prevalent infectious diseases worldwide which causes high morbidity and mortality. However, there is still limited understanding of the physiological processes that allow M. tuberculosis to survive in its host environment. One of the challenges is the limited availability of molecular probes that can be used to study some of the complex systems in mycobacteria. One such system is the ESX-3 secretion system, a specialized type VII secretion (T7S) system. This system is essential for optimal growth of pathogenic mycobacteria in low iron environments similar to that encountered by mycobacteria in macrophages during infection. EsxG, a protein of unknown function, is both encoded within the ESX-3 locus and secreted by the ESX-3 system.     

Labelling of live cells using fluorescent aptamers: binding reversal with DNA nucleases

A reversible cell labelling method has been developed for non-destructive and non-invasive cell labelling and purification. Our method uses high affinity single strand DNA (ssDNA) aptamers against surface exposed target molecules on cells. The aptamers are subsequently removed from the cell surface using DNase nuclease treatment. We exemplified our method by labelling human acute lymphoblastic leukemia cells with Qdot-ssDNA aptamers, and restoring them to the label-free condition by treatment with Benzonase. Binding of the fluorescent-aptamers to the cells was evaluated by measuring fluorescence intensity and was further confirmed using flow cytometry. Removal of the aptamers can be achieved in ~10 min by the DNase nuclease digestion. Incubation of cells with aptamers or with the nucleases results in no apparent damage to the cells and does not affect their growth rates. The latter were equivalent to the rates measured for the untreated cells. Our method provides an alternative to traditional antibody-based techniques and could be especially suitable for non-invasive reversible cell labelling and cell separations where maintaining native cell activity is needed.     

Isolation and characterization of bacteriophages specific for Campylobacter jejuni

Human infection by Campylobacter jejuni is mainly through the consumption of contaminated poultry products, which results in gastroenteritis and, rarely, bacteremia and polyneuropathies. In this study, six C. jejuni -specific bacteriophages (CPS1–6) were isolated by the spot-on-the-lawn technique from chicken samples in Korea and characterized for potential use as biocontrol agents. All isolated bacteriophages exhibited a high specificity, being able to lyse only C. jejuni , but not other Gram–negative bacteria, including C. coli , Escherichia coli , Salmonella spp., and Gram–positive bacteria. Bacteriophages contain an icosahedral head and a contractile tail sheath in transmission electron microscopy, and possess ds-DNA with an average genome size of approximately 145 kb; therefore, all bacteriophages are categorized into the Myoviridae family. Bacterial lysis studies in liquid media revealed that CPS2 could be used to control the growth of C. jejuni .     

Expression and characterization of cell-signalling molecules in Campylobacter jejuni

Aims: This study investigated the production and effects of cell‐signalling compounds on selected survival and virulence mechanisms of Campylobacter jejuni. Methods and Results: The production of Autoinducer 1 (AI‐1) compounds by Camp. jejuni was investigated in‐vitro using a variety of available AI‐1 bioassays. We further examined the role of a range of commercially available homoserine lactones (HSL) and a novel compound (cjA) isolated from Camp. jejuni. The selected attributes included the transformation to a viable but nonculturable (VBNC) state, biofilm formation, interleukin 8 (IL‐8) stimulation in INT‐407 cells and virulence gene expression as determined by qRT‐PCR. This study is the first to report an HSL or HSL mimic produced by Camp. jejuni. Short chained HSLs and the novel compound cjA prolonged the delay to a VBNC state as well as inhibiting biofilm formation and the majority of HSLs examined and the HSL mimic cjA significantly affected virulence gene expression as well as increasing the production of IL‐8 in challenged INT‐407 cells. Conclusions: Despite the lack of a homologous HSL kinase or sensor, Camp. jejuni appears to produce, as well as detect, exogenous signalling molecules and respond accordingly to aid in the survival and virulence capabilities of this micro‐organism. Significance and Impact of the Study: This study suggests that Camp. jejuni is able to detect and utilize as well as possibly produce cell‐signalling molecules that enhance both survival and virulence attributes. This possibility opens a new field in the search for Camp. jejuni reduction and elimination strategies.     

Minimal primer and primer-free SELEX protocols for selection of aptamers from random DNA libraries

Standard systematic evolution of ligands by exponential enrichment (SELEX) protocols require libraries that contain two primers, one on each side of a central random domain, which allow amplification of target-bound sequences via PCR or RT-PCR. However, these primer sequences cause nonspecific binding by their nature (generally adding about 20 nt on each end of the random sequence of about 30-40 nt), and can result in large numbers of false-positive binding sequences and/or interfere with good binding random sequences. Here, we have developed two DNA-based methods that reduce and/or eliminate the primer sequences from the target-binding step, thus reducing or eliminating the interference caused by the primer sequences. In these methods, the starting selection libraries contain a central random sequence that is: (i) flanked by only 2 nt on each side (minimal primer); or (ii) flanked only by either a 2- or 0-nt overhand on the 3' end (primer-free). These methods allow primer regeneration and re-elimination after and before selection, are fast and simple, and don't require any chemical modifications for selection in a variety of conditions. Further, the selection rounds are performed with DNA oligomers, which are generally employed as end product aptamers.     

Selection of DNA aptamers for breast cancer

A method of selection of DNA aptamers to breast tumor tissue based on the use of postoperative material has been developed. Breast cancer tissues were used as the positive target; the negative targets included benign tumor tissue, adjacent healthy tissues, breast tissues from mastopathy patients, and also tissues of other types of malignant tumors. During selection a pool of DNA aptamers demonstrating selective binding to breast cancer cells and tissues and insignificant binding to breast benign tissues has been obtained. These DNA aptamers can be used for identification of protein markers, breast cancer diagnostics, and targeted delivery of anticancer drugs.     

Molecular characterization of a Campylobacter jejuni lipoprotein with homology to periplasmic siderophore-binding proteins.

A genomic library of Campylobacter jejuni (NCTC 11351) was used to identify genes which could confer a hemolytic phenotype to Escherichia coli. Accordingly, when transformants were screened on blood plates, hemolytic colonies appeared at a frequency of 3 x 10(-4). The gene conferring the hemolytic activity was identified by subcloning and was found to be responsible for the phenotype of all hemolytic transformants isolated. The open reading frame conferring this activity encodes a protein of 36,244 Da with a typical endopeptidase type II leader sequence. The protein is modified with palmitic acid when it is processed in E. coli, confirming that it is a typical lipoprotein. The deduced gene product of 329 amino acids has significant homology to the group of solute binding proteins from periplasmic-binding-protein-dependent transport systems for ferric siderophores, including the FatB protein from Vibrio anguillarium and the FhuD protein from Bacillus subtilis. In particular, the protein contained the signature sequence for siderophore-binding proteins, suggesting that the protein may be the siderophore-binding protein component of an iron acquisition system of C. jejuni.     

Selection of DNA aptamers that bind to four organophosphorus pesticides

Single-stranded DNA (ssDNA) aptamers against four organophosphorus pesticides (phorate, profenofos, isocarbophos and omethoate) were simultaneously isolated from an immobilized random ssDNA library by systematic evolution of ligands by exponential enrichment (SELEX) technique. After 12 rounds of in vitro selection, five ssDNA aptamer candidates were selected and their binding affinities were identified by a novel method using a molecular beacon. Two of the five ssDNA sequences, SS2-55 and SS4-54, demonstrated higher affinities and specificities to the four organophosphorus pesticides. They were defined as broad-spectrum aptamers binding to four different targets and their simulated secondary structures showed highly distinct features with typical stem and loop structures. The dissociation constant of SS2-55 and SS4-54 binding to the four organophosphorus pesticides ranged from 0.8 to 2.5?μM. These aptamers offered application potential in the analysis and/or neutralization of the residues of the four organophosphorus pesticides.     

Cloning and characterization of the lytB gene of Campylobacter jejuni

LytB of Escherichia coli is an essential gene involved in penicillin tolerance and the stringent response. The lytB gene of Campylobacter jejuni was cloned and characterized. It could complement a temperature-sensitive E. coli lytB mutant. The C. jejuni lytB gene encodes a protein of 277 amino acids that has 34, 36 and 40% amino acid identity with the LytB proteins of E. coli, Haemophilus influenzae, and Synechocystis sp. PCC6803, respectively. The lytB gene is situated between the aroA gene and a gene that encodes ribosomal protein S1.     

Isolation and characterization of the flagellar hook of Campylobacter jejuni

Abstract A method for purification of the flagellar hook of Campylobacter jejuni is described. The hook was shown to be composed of a subunit protein, which has a molecular mass of 92,000 and an isoelectric point of pI 4.8. A monoclonal antibody and a polyvalent antiserum was raised against the purified flagellar hook of C. jejuni . Immuno-electronmicroscopy revealed that the epitope recognized by the monoclonal antibody is surface-located. However, this antibody reacted only with the hook of the immunization strain, but not with other strains or other flagellated bacteria. Thus, our data indicate that the immunodominant epitopes are located on the surface of the hook and that these epitopes are strain-specific.