Non-natural protein-protein communication mediated by a DNA-based, antibody-responsive device

We report here the rational design and optimization of an antibody responsive, DNA-based device that enables communication between pairs of otherwise non-interacting proteins. The device is designed to recognize and bind a specific antibody and, in response, undergo a conformational change that leads to the release of a DNA strand, termed the “translator,” that regulates the activity of a downstream target protein. As proof of principle, we demonstrate antibody-induced control of the proteins thrombin and Taq DNA polymerase. The resulting strategy is versatile and, in principle, can be easily adapted to control artificial protein-protein communication in artificial regulatory networks

Development of rapid methods for determination of Salmonella in meat products

The aim of the present study was the evaluation of two different techniques to detect Salmonella in meat: an ELISA assay using electrochemical detection coupled with a FIA system and a PCR method. A sandwich format was chosen for ELISA assay, using monoclonal and polyclonal antibodies against salmonella. A 35-cycle PCR was carried out for the research of genomic fragment. The assays were used to analyse samples of pork, chicken and bovine meat experimentally contaminated with different concentrations of S. Enteritidis. Results show that both methods were efficient, sensitive and rapid. After only 4 hours of incubation in pre-enrichment broth (buffered peptone water) it was possible to detect salmonella in meat experimentally contaminated with low concentrations (1-10 cells in 25 g) both with the ELISA assay and the PCR method

Molecular characterization of Yersinia enterocolitica strains to evaluate virulence associated genes

Introduction. Yersinia enterocolitica (Ye) species is divided into 6 biotypes (BT), 1A, 1B, 2, 3, 4, 5 classified based on biochemical reactions and about 70 serotypes, classified based on the structure of the lipopolysaccharide O-antigen. The BT1A is considered non-pathogenic, while the BT 1B-5 are considered pathogenic. Methods. Evaluate the distribution of eleven chromosomal and plasmid virulence genes, ail, ystA, ystB, myfA, hreP, fes, fepD, ymoA, sat, virF and yadA, in 87 Ye strains isolated from food, animals and humans, using two SYBR Green real-time PCR platforms. Results. The main results showed the presence of the ail and ystA genes in all the pathogenic bioserotypes analyzed. The ystB, on the other hand, was identified in all non-pathogenic strains biotype 1A. The target fes, fepD, sat and hreP were found in both pathogenic biotypes and in BT1A strains. The myfA gene was found in all pathogenic biotype and in some Ye BT1A strains. The virF and yadA plasmid genes were mainly detected in bioserotype 4/O:3 and 2/O:9, while ymoA was identified in all strains. Conclusions. The two molecular platforms could be used to better define some specific molecular targets for the characterization and rapid detection of Ye in different sources which important implications for food safety and animal and human health

First isolation of Salmonella enterica serovar Napoli from wild birds in Italy

AbstractSalmonella enterica serovar Napoli (S. Napoli) is an emerging serovar in Italy. It accounts for 2-4% of all serovars isolated from human infections. The zoonotic origin of this serovar is still unknown and this makes difficult to apply any control intervention. We report here the isolation of S. Napoli from a river nightingale (Cettia cetti, Temminck 1820) which represents the first description of this serovar from wild birds. This finding adds knowledge to the ecology of S. Napoli and addresses further studies aimed to assess the epidemiologic link between S. Napoli isolated from wild birds, food, environmental sources and human infections.

Development of rapid methods for determination of Salmonella in meat products

The aim of the present study was the evaluation of two different techniques to detect Salmonella in meat: an ELISA assay using electrochemical detection coupled with a FIA system and a PCR method. A sandwich format was chosen for ELISA assay, using monoclonal and polyclonal antibodies against salmonella. A 35-cycle PCR was carried out for the research of genomic fragment. The assays were used to analyse samples of pork, chicken and bovine meat experimentally contaminated with different concentrations of S. Enteritidis. Results show that both methods were efficient, sensitive and rapid. After only 4 hours of incubation in pre-enrichment broth (buffered peptone water) it was possible to detect salmonella in meat experimentally contaminated with low concentrations (1-10 cells in 25 g) both with the ELISA assay and the PCR method.</p

Electrochemical Biosensors for Rapid Detection of Foodborne Salmonella: A Critical Overview

Abstract: Salmonella has represented the most common and primary cause of food poisoning in many countries for at least over 100 years. Its detection is still primarily based on traditional microbiological culture methods which are labor-intensive, extremely time consuming, and not suitable for testing a large number of samples. Accordingly, great efforts to develop rapid, sensitive and specific methods, easy to use, and suitable for multi-sample analysis, have been made and continue. Biosensor-based technology has all the potentialities to meet these requirements. In this paper, we review the features of the electrochemical immunosensors, genosensors, aptasensors and phagosensors developed in the last five years for Salmonella detection, focusing on the critical aspects of their application in food analysis

Comparison of PCR, Electrochemical Enzyme-Linked Immunosorbent Assays, and the Standard Culture Method for Detecting Salmonella in Meat Products

An electrochemical enzyme-linked immunosorbent assay (ELISA) coupled with flow injection analysis (ELISA-FIA) and a PCR-based method using ST11 and ST15 primers for detecting salmonellae in meat were evaluated in comparison with the International Organization for Standardization (ISO) culture method. The methods were applied to experimentally contaminated and naturally contaminated meat samples. The results showed that both ELISA-FIA and PCR allowed detection of salmonella in a product contaminated with a low number of the microorganisms (1 to 10 salmonellae/25 g) after only 5 h of incubation of preenrichment broth, and they were just as effective as the ISO method

SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A▿

Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 101 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%)