Selection of spore-specific aptamers for Geobacillus stearothermophilus, a food spoilage bacterium.

Due to their ability to form extremely heat resistant spores, anaerobic bacteria are responsible for frequent food spoilage. The development of rapid and specific methods for the detection and quantification of spore contamination is therefore of major interest. In this paper, we describe for the first time the selection of aptamers specific to spores of Geobacillus stearothermophilus (Gbs), which induce flat sour spoilage in vegetable cans. Eighteen Spore-SELEX cycles were performed including 4 counter-selections with 12 bacteria commonly found in cannery. To optimise candidate amplification, PCR in emulsion was performed, and high-throughput sequencing analysis was applied to follow candidate evolution. Sequencing of aptamers from cycle 18 revealed 43 overrepresented sequences whose copy number exceeds 0.15% of the total obtained sequences. Within this group, the A01 aptamer presented a much higher enrichment with a relative abundance of 17.71%. Affinity and specificity for Gbs spores of the 10 most abundant candidates at cycle 18 were confirmed by PCR assay based on aptamer-spore complex formation and filtration step. Obtaining these aptamers is the starting point for the future development of biosensors dedicated to the detection of Gbs spores.

[Sources and fate of pathogenic microorganisms in aquatic environments]. / Sources et devenir des micro-organismes pathogènes dans les environnements aquatiques.

A large number of human waterborne infections are caused by microorganisms including viruses, bacteria or protozoa. These microorganisms are either naturally presents in aquatic environments, or come from fecal sources. They may stay in aquatic ecosystems for a long time before contaminating a new host. Aquatic environments are subjected to high variability of physico- chemical parameters. They include a microbial community that is more or less adapted to these environmental changes. Thus, numerous interactions occur between microorganisms, which are pathogenic or not, and most of them are still unknown. This review focused on interactions between pathogen microorganisms and their environment. More specifically we addressed the question of the diversity of the sources of contamination, their contribution to the microbiological pollution of the aquatic environment, and the ability of some pathogens to survive adverse environmental conditions.

Development of an electrochemical biosensor for the detection of aflatoxin M1 in milk.

We have developed an electrochemical immunosensor for the detection of ultratrace amounts of aflatoxin M(1) (AFM(1)) in food products. The sensor was based on a competitive immunoassay using horseradish peroxidase (HRP) as a tag. Magnetic nanoparticles coated with antibody (anti-AFM(1)) were used to separate the bound and unbound fractions. The samples containing AFM(1) were incubated with a fixed amount of antibody and tracer [AFM(1) linked to HRP (conjugate)] until the system reached equilibrium. Competition occurs between the antigen (AFM(1)) and the conjugate for the antibody. Then, the mixture was deposited on the surface of screen-printed carbon electrodes, and the mediator [5-methylphenazinium methyl sulphate (MPMS)] was added. The enzymatic response was measured amperometrically. A standard range (0, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.3, 0.4 and 0.5 ppb) of AFM(1)-contaminated milk from the ELISA kit was used to obtain a standard curve for AFM(1). To test the detection sensitivity of our sensor, samples of commercial milk were supplemented at 0.01, 0.025, 0.05 or 0.1 ppb with AFM(1). Our immunosensor has a low detection limit (0.01 ppb), which is under the recommended level of AFM(1) [0.05 µg L-1 (ppb)], and has good reproducibility.

Detection of Salmonella in Food Matrices, from Conventional Methods to Recent Aptamer-Sensing Technologies.

Rapid detection of the foodborne pathogen Salmonella in food processing is of crucial importance to prevent food outbreaks and to ensure consumer safety. Detection and quantification of Salmonella species in food samples is routinely performed using conventional culture-based techniques, which are labor intensive, involve well-trained personnel, and are unsuitable for on-site and high-throughput analysis. To overcome these drawbacks, many research teams have developed alternative methods like biosensors, and more particularly aptasensors, were a nucleic acid is used as biorecognition element. The increasing interest in these devices is related to their high specificity, convenience, and relative rapid response. This review aims to present the advances made in these last years in the development of biosensors for the detection and the quantification of Salmonella, highlighting applications on meat from the chicken food chain.

Selection of DNA aptamers against penicillin G using Capture-SELEX for the development of an impedimetric sensor.

This paper describes for the first time the selection of aptamers selective to penicillin. Aptamers were selected using a specific process called Capture-SELEX (Systematic Evolution of Ligands by Exponential Enrichment). This technique is based on the selection of DNA aptamers using penicillin G in solution while the ssDNA library is fixed on a support. One aptamer showing a good affinity to penicillin was finally selected and tested in electrochemical sensor configuration, using electrochemical impedance spectroscopy as detection technique. The developed aptasensor allowed the detection of penicillin in a wide concentration range, comprised between 0.4 and 1000µgL-1 Such performance was compatible with milk analysis, as the maximum residue limit tolerated in this matrix is 4µgL-1. The selectivity of the developed sensor was also studied, showing that the sensor was also able to bind other beta-lactam antibiotics, although with a weaker affinity. Finally the sensor was used for detection of penicillin G in milk. It was shown that a simple sample treatment with isopropanol followed by filtration was sufficient to eliminate matrix effects, allowing the determination of penicillin in milk at concentrations compatible with legislation requirements.

Development of an impedimetric aptasensor for the determination of aflatoxin M1 in milk.

An aptasensor was designed for the determination of aflatoxin M1 (AFM1) in milk based on DNA-aptamer recognition and electrochemical impedance spectroscopy detection. A hexaethyleneglycol-modified 21-mer oligonucleotide was immobilized on a carbon screen-printed electrode through carbodiimide immobilization, after diazonium activation of the sensing surface. Cyclic voltammetry and electrochemical impedance spectroscopy in the presence of ferri/ferrocyanide redox probe were used to characterize each step of the aptasensor development. Aptamer-AFM1 interaction induced an increase in electron-transfer resistance, allowing the determination of AFM1 in buffer in the range 2-150 ng/L (LOD=1.15 ng/L). Application to milk analysis showed that a preliminary treatment was mandatory. A simple filtration through a 0.2 µm PTFE membrane allowed determination of AFM1 in milk for concentrations ranging from 20 to 1000 ng/kg. These performances are compatible with the AFM1 levels set in European Union for milk and dairy products for adults (50 ng/kg) and infants (25 ng/kg).

Colorimetric and electrochemical genosensors for the detection of Escherichia coli DNA without amplification in seawater.

Monitoring seawater, particularly recreational water, for indicator bacteria presence is required to protect the public from exposure to fecal pollution and to guarantee the safety of the swimming areas. Two methods for the detection and quantification of Escherichia coli DNA were developed: a colorimetric assay in a microplate and an electrochemical biosensor. These assays were based on the double hybridization recognition of a single-strand DNA capture probe immobilized onto the microplate or the screen-printed carbon electrode to its complementary ssDNA, which is hybridized with an ssDNA signal probe labeled with horseradish peroxidase enzyme. The hybridization recognition step used the colorimetric monitoring of the oxidation state of the 3,3',5,5'-tetramethylbenzidine. The electrochemical monitoring of the oxidation state of 5 methyl-phenazinium methyl sulfate was allowed when the horseradish-peroxidase was in the presence of the mediator (5 methyl-phenazinium methyl sulfate and hydrogen peroxide). These approaches allow for the detection and quantification of 10(2) to 10(3) cells of E. coli in 5l of seawater samples in less than 5h. Detection was achieved without a nucleic acid amplification step. The specificity of the two methods against E. coli was demonstrated by testing a panel of bacteria. The two methods can be used for on-site monitoring of seawater quality.