DNA Capture Probes Research Articles

Visual DNA microarray for detection of epidermal growth factor receptor (EGFR) gene mutations

Objective. To develop a gold nanoparticle-based visual DNA microarray for simple and rapid screening of EGFR gene mutations. Methods. The DNA fragments contain epidermal growth factor receptor (EGFR) exons 18, 19, 20 and 21 were amplified by polymerase chain reaction (PCR) using biotin-modified primers. The amino-modified oligonucleotides were immobilized on glass surface, which were used as the capturing probes to bind the complement biotinylated target DNA. After the PCR product has been hybridized to the immobilized capture probe DNA on the glass slides, the Streptavidin-conjugated gold nanoparticles were introduced to the microarray via specific binding to 5ʹ-end biotin of the PCR products. The hybridization signal on array spots was enhanced and visualized by silver amplification. The EGFR mutation in 286 clinical samples from cancer patients were tested using the gold nanoparticle-based microarray and verified with Sanger DNA sequencing method. Results. A novel visual DNA microarray has been developed to detect EGFR mutations in tumor tissue specimens rapidly; its limit of detection (LOD) is up to 10−9 mol/L and distinguishes power to detect 5% gene mutation in the mixture samples. Conclusion. For its high specificity and sensitivity, simplicity, lower price and higher speed, the present visual mutation detecting technique has potential application in clinical fields.

Electrogenerated chemiluminescence biosensing method for methyltransferase activity using tris(1, 10-phenanthroline) ruthenium-assembled graphene oxide

A novel electrogenerated chemiluminescence (ECL) biosensing method for highly sensitive detection of DNA methylation and assay of the methyltransferase (MTase) activity was developed by using the methylation-sensitive restriction endonuclease Dpn I and methylation-responsive hairpin-capture DNA probe to improve selectivity and by employing signal amplification of graphene oxide (GO) to enhance the assay sensitivity. The ECL biosensing electrode was fabricated by self-assembling a design of 5′-thiol modified methylation-responsive hairpin-capture DNA probe on the surface of a gold electrode. When DNA adenine methylation (Dam) MTase and S-adenosylmethionine were introduced, all adenines in the symmetric tetranucleotide 5′-GATC-3′ of hairpin-capture DNA probe on the biosensing electrode were methylated. After the methylated biosensing electrode was treated by Dpn I endonuclease, the methylated adenines were cleaved, methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the “capture DNA probe” section on the gold electrode. Subsequently, the remained “capture DNA probe” on the gold electrode can hybridize with the 5′-amino modified DNA probe. Finally, tris(1, 10-phenanthroline) ruthenium (Ru(phen)32+)-assembled GO composites were conjugated to the electrode surface via EDC–NHS coupling, a strong ECL response was electrochemically generated. The increased ECL intensity was proportion to Dam MTase activity in the range from 0.05U/mL to 40U/mL with a detection limit of 0.02U/mL. The present work demonstrates that the combination of the enzyme-linkage and GO as a platform for signal probe is a great promising approach for MTase activity and evaluation of the capability of inhibitors for the MTase.

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III-aided cycling amplification

A novel magneto-DNA duplex probe for bacterial DNA detection based on exonuclease III (Exo-III) aided cycling amplification has been developed. This magneto-DNA duplex probe contains a partly hybrid fluorophore-modified capture probe and a fluorophore-modified signal probe with magnetic microparticle as carrier. In the presence of a perfectly matched target bacterial DNA, blunt 3′-terminus of the capture probe is formed, activating the Exo-III aided cycling amplification. Thus, Exo-III catalyzes the stepwise removal of mononucleotides from this terminus, releasing both fluorophore-modified signal probe, fluorescent dyes of the capture probe and target DNA. The released target DNA then starts a new cycle, while released fluorescent fragments are recovered with magnetic separation for fluorescence signal collection. This system exhibited sensitive detection of bacterial DNA, with a detection limit of 14pM because of the unique cleavage function of Exo-III, high fluorescence intensity, and separating function of magneto-DNA duplex probes. Besides this sensitivity, this strategy exhibited excellent selectivity with mismatched bacterial DNA targets and other bacterial species targets and good applicability in real seawater samples, hence, this strategy could be potentially used for qualitative and quantitative analysis of bacteria.

One-pot synthesis of GO/AgNPs/luminol composites with electrochemiluminescence activity for sensitive detection of DNA methyltransferase activity

DNA methyltransferases catalyze the transfer of a methyl group from S-adenosylmethionine to the target adenine or cytosine, eventually inducing the DNA methylation in both prokaryotes and eukaryotes. Herein, we developed a novel electrochemiluminescence biosensor to quantify DNA adenine methylation (Dam) methyltransferase (MTase) employing signal amplification of GO/AgNPs/luminol composites to enhance the assay sensitivity. The method was developed by designing a capture probe DNA, which was immobilized on gold electrode surface, to hybridize with azide complementary DNA to form the azide-terminated dsDNA. Then, alkynyl functionalized GO/AgNPs/luminol composites as the signal probe were immobilized to azide-terminated dsDNA modified electrode via click chemistry, resulting in a high electrochemiluminescence (ECL) signal. Once the DNA hybrid was methylated (under catalysis of Dam MTase) and further cleaved by Dpn I endonuclease (a site-specific endonuclease recognizing the duplex symmetrical sequence of 5′-G-Am-T-C-3′), GO/AgNPs/luminol composites release from the electrode surface to the solution, leading to significant reduction of the ECL signal. The change of the ECL intensity is related to the methylation status and MTase activity, which forms the basis of MTase activity assay and site-specific methylation determination. This novel strategy can be further used as a universal method for other transferase determination by designing various transferase-specific DNA sequences. In addition, this method can be used for the screening of antimicrobial drugs and has a great potential to be further applied in early clinical diagnosis.

Electrical potential-assisted DNA hybridization. How to mitigate electrostatics for surface DNA hybridization.

Surface-confined DNA hybridization reactions are sensitive to the number and identity of DNA capture probes and experimental conditions such as the nature and the ionic strength of the electrolyte solution. When the surface probe density is high or the concentration of bulk ions is much lower than the concentration of ions within the DNA layer, hybridization is significantly slowed down or does not proceed at all. However, high-density DNA monolayers are attractive for designing high-sensitivity DNA sensors. Thus, circumventing sluggish DNA hybridization on such interfaces allows a high surface concentration of target DNA and improved signal/noise ratio. We present potential-assisted hybridization as a strategy in which an external voltage is applied to the ssDNA-modified interface during the hybridization process. Results show that a significant enhancement of hybridization can be achieved using this approach.

Integrated air stream micromixer for performing bioanalytical assays on a plastic chip.

This paper describes the design, functioning and use of an integrated mixer that relies on air flux to agitate microliter entities of fluid in an embedded microfluidic cavity. The system was fabricated from multiple layers of a thermoplastic elastomer and features circuits for both liquid and air supply along with pneumatic valves for process control. Internally-dyed polymer particles have been used to visualize flow within the fluid phase during agitation. Numerical modelling of the micromixer revealed an overall efficacy of 10(-1) to 10(-2) for momentum transfer at the air-water interface. Simulation of air vortex dynamics showed dependency of the flow pattern on the velocity of the flux entering the cavity. Three bioanalytical assays have been performed as proof-of-concept demonstrations. In a first assay, cells of Listeria monocytogenes were combined with magnetic nanoparticles (NPs), resulting in high-density coverage of the bacteria's surface with NPs after 1 min of agitation. This finding is contrasted by a control experiment without agitation for which interaction between bacteria and NPs remains low. In a second one, capture and release of genomic DNA from fungi through adsorption onto magnetic beads was tested and shown to be improved by agitation compared to non-agitated controls. A third assay finally involved fluorescently-labelled target oligonucleotide strands and polystyrene particles modified with DNA capture probes to perform detection of nucleic acids on beads. Excellent selectivity was obtained in a competitive hybridization process using a multiplexed micromixer chip design.

Ultrasensitive and selective signal-on electrochemical DNA detection via exonuclease III catalysis and hybridization chain reaction amplification

This work reported a novel, ultrasensitive, and selective platform for electrochemical detection of DNA, employing an integration of exonuclease III (Exo-III) assisted target recycling and hybridization chain reaction (HCR) for the dual signal amplification strategy. The hairpin capture probe DNA (C-DNA) with an Exo-III 3′ overhang end was self-assembled on a gold electrode. In the presence of target DNA (T-DNA), C-DNA hybridized with the T‐DNA to form a duplex region, exposing its 5′ complementary sequence (initiator). Exo-III was applied to selectively digest duplex region from its 3-hydroxyl termini until the duplex was fully consumed, leaving the remnant initiator. The intact T-DNA spontaneously dissociated from the structure and then initiated the next hybridization process as a result of catalysis of the Exo-III. HCR event was triggered by the initiator and two hairpin helper signal probes labeled with methylene blue, facilitating the polymerization of oligonucleotides into a long nicked dsDNA molecule. The numerous exposed remnant initiators can trigger more HCR events. Because of integration of dual signal amplification and the specific HCR process reaction, the resultant sensor showed a high sensitivity for the detection of the target DNA in a linear range from 1.0fM to 1.0nM, and a detection limit as low as 0.2fM. The proposed dual signal amplification strategy provides a powerful tool for detecting different sequences of target DNA by changing the sequence of capture probe and signal probes, holding a great potential for early diagnosis in gene-related diseases.

Highly sensitive electrochemical sensor using a MWCNTs/GNPs-modified electrode for lead (II) detection based on Pb(2+)-induced G-rich DNA conformation.

A sensitive electrochemical lead ion (Pb(2+)) sensor based on carboxylic acid group functionalized multi-walled carbon nanotubes (MWNTs-COOH) and direct electrodeposited gold nanoparticles (GNPs) was developed for Pb(2+) detection. The DNA capture probe was self-assembled onto the surface of the modified electrode for hybridizing with the guanine-rich (G-rich) aptamer probe and for forming the DNA double helix structure. When Pb(2+) was added in, the DNA duplex unwound and formed a stabilized G-quadruplex (G4) due to the Pb(2+)-induced G-rich DNA conformation. Also, methylene blue (MB) was selected as the G4-binding indicator. Compared with previous Pb(2+) sensors, the proposed sensor had better sensitivity, because the modified MWCNTs/GNPs could provide a large surface area and good charge-transport capacity to dramatically improve the DNA attachment quantity and sensor performance. The sensor could detect Pb(2+) in a range from 5.0 × 10(-11) to 1.0 × 10(-14) M, with a detection of 4.3 × 10(-15) M.

Sensitive colorimetric biosensing for methylation analysis of p16/CDKN2 promoter with hyperbranched rolling circle amplification

A simple, fast and sensitive colorimetric biosensing method for DNA methylation analysis was developed by combining methylation-sensitive endonuclease based digestion with hyperbranched rolling circle amplification (HRCA) induced signal enhancement. The assay was carried out on a DNA capture probe modified 96-cell microplate with four sequential steps of target recognition, endonuclease-based digestion, isothermal HRCA, and enzyme-catalyzed colorimetric readout within 3h. The semi-quantitative and precise analysis of methylated DNA could be easily achieved by naked eye and absorbance measurements, respectively. The strategy exhibited excellent detection specificity and accuracy with a log-linear response to methylated DNA from 100fM to 10nM. As a proof of concept, the assay was applied to investigate the methylation status of p16/CDKN2 promoter of breast cancer patients. The methylated p16 concentration was not significantly associated with the clinical parameters. The proposed method allowed efficient methylation detection with simplicity, rapidness, low cost and high sensitivity, showing great promise for application in early diagnosis of methylation-related diseases.

Label-free and amplified electrogenerated chemiluminescence biosensing method for the determination of DNA methyltransferase activity using signal reagent-assembled graphene oxide

A novel label-free electrogenerated chemiluminescence (ECL) biosensing method for the determination of DNA methyltransferase (MTase) activity was developed on base of enzyme-linkage reactions and tris(1, 10-phenanthroline) ruthenium-assembled graphene oxide (GO) served as an ECL signal compound. The ECL biosensing electrode was fabricated by self-assembling 5′-thiol modified hairpin-capture DNA probe containing methylation recognition site 5′-GATC-3′ on the surface of a gold electrode. When DNA adenine methylation (Dam) MTase and S-adenosyl-L-methionine were introduced, all adenine residues within 5′-GATC-3′ of hairpin-capture DNA probe on the biosensing electrode were methylated. After the methylated biosensing electrode was treated by the methylation-sensitive restriction endonuclease Dpn I, the methylated adenines were cleaved, methylation-induced scission of hairpin-capture DNA probe would displace the hairpin section and remain the “capture DNA probe” section on the gold electrode, then a long ssDNA was immobilized via the partial hybridization reaction between long ssDNA and hairpin-capture DNA probe remained section, the more binding site allow tris(1, 10-phenanthroline) ruthenium-assembled GO to be more bound to the long ssDNA on the electrode surface through both hydrophobic and π–π stacking interaction, in conjunction with the generation of a increased ECL signal. The ECL intensity versus the concentration of Dam MTase was linear in the range from 0.02 unit/mL to 10 unit/mL. The detection limit was 0.01 unit/mL. This work demonstrates that using the different affinities of GO for ssDNA and dsDNA for the fabrication of the label-free ECL biosensing method for DNA MTase activity is promising approach.

Tunable stringency aptamer selection and gold nanoparticle assay for detection of cortisol.

The first-known aptamer for the stress biomarker cortisol was selected using a tunable stringency magnetic bead selection strategy. The capture DNA probe immobilized on the beads was systematically lengthened to increase the number of bases bound to the complementary pool primer regions following selection enrichment. This resulted in a single sequence (15-1) dominating the final round 15 pool, where the same sequence was the second-highest copy number candidate in the enriched pool with the shorter capture DNA probe (round 13). A thorough analysis of the next-generation sequencing results showed that a high copy number may only correlate with enhanced affinity under certain stringency and enrichment conditions, in contrast with prior published reports. Aptamer 15-1 demonstrated enhanced binding to cortisol (K(d) = 6.9 ± 2.8 μM by equilibrium dialysis; 16.1 ± 0.6 μM by microscale thermophoresis) when compared with the top sequence from round 13 and the negative control progesterone. Whereas most aptamer selections terminate at the selection round demonstrating the highest enrichment, this work shows that extending the selection with higher stringency conditions leads to lower amounts eluted by the target but higher copy numbers of a sequence with enhanced binding. The structure-switching aptamer was applied to a gold nanoparticle assay in buffer and was shown to discriminate between cortisol and two other stress biomarkers, norepinephrine and epinephrine, and a structurally analogous biomarker of liver dysfunction, cholic acid. We believe this approach enhances aptamer selection and serves as proof-of-principle work toward development of point-of-care diagnostics for medical, combat, or bioterrorism targets.

Label-free and non-enzymatic detection of DNA based on hybridization chain reaction amplification and dsDNA-templated copper nanoparticles

A label-free and non-enzymatic amplification fluorescent method for detection of DNA has been developed by using hybridization chain reaction (HCR) and dsDNA-templated copper nanoparticles (CuNPs). First, the biotinylated capture DNA probes were immobilized on the streptavidin-modified beads through the interaction of biotin and streptavidin. Then, target DNA hybridized with the capture DNA probes, which formed a hybridized DNA with sticky end. The sticky end triggered the HCR process and formation of dsDNA polymers while two hairpin probes coexisted. Subsequently, the dsDNA polymers were employed as template for synthesis of CuNPs with excellent fluorescent properties, which provided a label-free, non-enzymatic signal response. Meanwhile, the fluorescence sensing depended on the target DNA triggered HCR, which render this method a high selectivity against single-base mismatch sequences. The concept and methodology developed in this work show great promise in the quantitative detection of DNA in biological and medical applications.

Ratiometric electrochemical proximity assay for sensitive one-step protein detection.

This work proposes the concept of ratiometric electrochemical proximity assay (REPA), which can be used for one-step, highly sensitive and selective detection of protein. The assay strategy was achieved on a sensing interface that was formed by hybridization of methylene blue (MB)-labeled antibody-DNA probe (MB-DNA1-Ab1) with ferrocene (Fc)-labeled DNA capture probe (Fc-P) modified gold electrode. On the interface the target protein could trigger the formation of immunocomplex between MB-DNA1-Ab1 and detection antibody-DNA probe (Ab2-DNA2) and subsequently the proximity hybridization of DNA1-DNA2, which led to the departure of MB-DNA1-Ab1 from the interface. The remained Fc-P could form a hairpin structure to take Fc group to electrode surface. Therefore, the recognition of target protein to Ab1 and Ab2 resulted in both the “signal-off” of MB and the “signal-on” of Fc for dual-signal electrochemical ratiometric readout. The proposed REPA could be carried out in one-step with 40-min duration and showed a wide detection range from 0.05 to 100 ng/mL with pg/mL limit of detection, displaying great potential for convenient point-of-care testing and commercial application.

Femtomolar level detection of BRCA1 gene using a gold nanoparticle labeled sandwich type DNA sensor

We demonstrate the amplified detection of BRCAI gene based on the gold nanoparticle labeled DNA sensor. The sensor was based on a “sandwich” detection strategy, which involved an immobilized capture probe DNA (DNA-c), Target DNA (DNA-t) and gold nanoparticle conjugated reporter probe DNA (DNA-r.AuNP). The sensor surface was characterized by scanning electron microscopy (SEM) and scanning tunneling microscopy (STM). Detection capability of the sensor was studied with I–V measurements using either scanning tunneling microscopy (STM) or Keithley 2400 Source Meter SMU Instrument. The DNA sensor could detect up to 1fM DNA target (5.896fg of BRCA 1gene/ml) and exhibited excellent selectivity against noncomplementary sequences and three base mismatch complementary targets. Good reproducibility, high sensitivity, good stability and reusability of the developed sensor surface showed its application in early cancer diagnosis.

Aptamer-based microfluidic beads array sensor for simultaneous detection of multiple analytes employing multienzyme-linked nanoparticle amplification and quantum dots labels

This study reports the development of an aptamer-mediated microfluidic beads-based sensor for multiple analytes detection and quantification using multienzyme-linked nanoparticle amplification and quantum dots labels. Adenosine and cocaine were selected as the model analytes to validate the assay design based on strand displacement induced by target–aptamer complex. Microbeads functionalized with the aptamers and modified electron rich proteins were arrayed within a microfluidic channel and were connected with the horseradish peroxidases (HRP) and capture DNA probe derivative gold nanoparticles (AuNPs) via hybridization. The conformational transition of aptamer induced by target–aptamer complex contributes to the displacement of functionalized AuNPs and decreases the fluorescence signal of microbeads. In this approach, increased binding events of HRP on each nanosphere and enhanced mass transport capability inherent from microfluidics are integrated for enhancing the detection sensitivity of analytes. Based on the dual signal amplification strategy, the developed aptamer-based microfluidic bead array sensor could discriminate as low as 0.1pM of adenosine and 0.5pM cocaine, and showed a 500-fold increase in detection limit of adenosine compared to the off-chip test. The results proved the microfluidic-based method was a rapid and efficient system for aptamer-based targets assays (adenosine (0.1pM) and cocaine (0.5pM)), requiring only minimal (microliter) reagent use. This work demonstrated the successful application of aptamer-based microfluidic beads array sensor for detection of important molecules in biomedical fields.

Electrochemiluminescence bioassay for thrombin based on dynamic assembly of aptamer, thrombin and N-(aminobutyl)-N-(ethylisoluminol) functionalized gold nanoparticles

An electrochemiluminescence (ECL) bioassay was developed for the sensitive and selective detection of thrombin, based on dynamic interaction of aptamer, thrombin and N-(aminobutyl)-N-(ethylisoluminol) (ABEI) functionalized gold nanoparticles (ABEI-AuNPs). Gold nanoparticles by citrate reduction (AuNPs) were firstly assembled onto a gold electrode through 1, 3-propanedithiol, which was further connected with thiolated DNA capture probe. Then biotinylated DNA aptamer probe was assembled onto the modified electrode through the hybridization between capture probe and aptamer probe. After adding target thrombin, aptamer could bind tightly to target molecules to form a tertiary target–aptamer complex with a binding constant greater than DNA duplex, leading to partial extrication of biotinylated DNA aptamer probe from the surface of electrode. Finally, the ABEI-AuNPs coated with streptavidin (SA) were connected with the biotinylated DNA aptamer probe left after specific binding with thrombin to form the aptamer-ABEI-AuNPs complex on the electrode. When a double-step potential was applied to the electrode, an ECL signal was generated and recorded. The decrease of ECL signal was in proportion to the concentration of thrombin over the range of 1.0×10−12–1.0×10−9M with a detection limit of 3.8×10−13M. The proposed bioassay for the determination of thrombin is sensitive, specific, simple and fast. Finally, being challenged in real blood sample, the proposed bioassay was confirmed to be a good prospect for the detection of thrombin. This work provides a new way to design aptamer-based protocols for the determination of biologically important substances.

Simple diazonium chemistry to develop specific gene sensing platforms

A simple strategy for covalent immobilizing DNA sequences, based on the formation of stable diazonized conducting platforms, is described. The electrochemical reduction of 4-nitrobenzenediazonium salt onto screen-printed carbon electrodes (SPCE) in aqueous media gives rise to terminal grafted amino groups. The presence of primary aromatic amines allows the formation of diazonium cations capable to react with the amines present at the DNA capture probe. As a comparison a second strategy based on the binding of aminated DNA capture probes to the developed diazonized conducting platforms through a crosslinking agent was also employed. The resulting DNA sensing platforms were characterized by cyclic voltammetry, electrochemical impedance spectroscopy and spectroscopic ellipsometry. The hybridization event with the complementary sequence was detected using hexaamineruthenium (III) chloride as electrochemical indicator. Finally, they were applied to the analysis of a 145-bp sequence from the human gene MRP3, reaching a detection limit of 210pgμL−1.

PCR-Free Ultrasensitive Capacitive Biosensor for Selective Detection and Quantification of Enterobacteriacea DNA

This paper presents a flow-based ultrasensitive capacitive biosensor for the detection of bacterial DNA. The used sensor chip consists of a gold electrode, insulated with a polytyramine layer and covalently tagged with a DNA capture probe. The hybridization of target DNA to the capture probe resulted in sensor response. The sensor response was linear vs. log concentration in the range 1.0 × 10-12 to 1.0 × 10-7 moles per litre with a detection limit of 6.5 × 10-13 M. An alternative approach to bacterial DNA sample preparation for a flow-based analysis is also reported. The approach involved application of a thermostable ssDNA binding protein to prevent re-annealing of a heatdenatured target DNA prior to analysis. During analysis, formamide was integrated in the running buffer to denature ET SSB. E. coli DNA corresponding to 10 cells per millilitre of sample was detected in 15 min by this DNA-sensor. The sensor chip could be re-used up to 20 times with RSD of < 6%. The DNA-sensor chip was able to discriminate between Enterobacteriaceae (E. coli) and Lactobacillaceae (L. reuteri) DNAs. The reported DNA-sensor lays the groundwork for incorporating the method into an integrated system for in-field bacteria detection.

Highly improved specificity for hybridization-based microRNA detection by controlled surface dissociation

Poor specificity has been a lingering problem in many microRNA profiling methods, particularly surface hybridization-based methods such as microarrays. Here, we carefully investigated surface hybridization and dissociation processes of a number of sequentially similar microRNAs against nucleic acid capture probes. Single-base mismatched microRNAs were similarly hybridized to a complementary DNA capture probe and thereby poorly discriminated during conventional stringent hybridization. Interestingly, however, mismatched microRNAs showed significantly faster dissociation from the probe than the perfectly matched microRNA. Systematic analysis of various washing conditions clearly demonstrated that extremely high specificity can be obtained by releasing non-specific microRNAs from assay surfaces during a stringent and controlled dissociation step. For instance, compared with stringent hybridization, surface dissociation control provided up to 6-fold better specificity for Let-7a detection than for other Let-7 family microRNAs. In addition, a synthetically introduced single-base mismatch on miR206 was almost completely discriminated by optimized surface dissociation of captured microRNAs, while this mismatch was barely distinguished from target miR206 during stringent hybridization. Furthermore, a single dissociation condition was successfully used to simultaneously measure four different microRNAs with extremely high specificity using melting temperature-equalized capture probes. The present study on selective dissociation of surface bound microRNAs can be easily applied to various hybridization based detection methods for improved specificity.

Clostridium spp. discrimination with a simple bead-based fluorescence assay

C. chauvoei is the causative agent of blackleg, an endogenous bacterial infection which usually affects cattle and other ruminants. Due to the fact that the symptoms of this severe disease are very similar to the phenotype caused by an infection with C. septicum, a reliable differentiation of C. chauvoei from other Clostridium spp. is mandatory. Traditional microbiological detection methods are time consuming and the proper specification is hampered by the overgrowing tendency of swarming C. septicum colonies when both species are in the clinical sample. Thus, there is an urgent need to improve and simplify the specific detection of C. chauvoei and C. septicum. We report an easy and fast Clostridium spp. discrimination method via a magnetic bead-based fluorescence assay. To that end, the target DNA was amplified using 16S-23S rDNA spacer region specific primers. These PCR products were employed to generate single-stranded capture probe DNA, which was immobilized on magnetic beads. Functionalized magnetic particles exhibit numerous advantages, like their simple manipulation in combination with a huge binding capacity of biomolecules and make therefore excellent biosensors. In this context, the discrimination between C. chauvoei and C. septicum was realized by means of hybridization with complementary detection probe DNA. Finally, fluorescence spectroscopy allowed the signal readout. With this approach a precise discrimination between C. chauvoei, C. septicum and C. carnis was accomplished.