Rapid detection of Pseudomonas aeruginosa using a DNAzyme-based sensor.

In the present study, a DNAzyme was screened in vitro through the use of a DNA library and crude extracellular mixture (CEM) of Pseudomonas aeruginosa. Following eight rounds of selection, a DNAzyme termed PAE-1 was obtained, which displayed high rates of cleavage with strong specificity. A fluorescent biosensor was designed for the detection of P. aeruginosa in combination with the DNAzyme. A detection limit as low as 1.2 cfu/ml was observed. Using proteases and filtration, it was determined that the target was a protein with a molecular weight of 10 kDa-50 kDa. The DNAzyme was combined with a polystyrene board to construct a simple indicator plate sensor which produced a color that identified the target within 10 min. The results were reliable when tap water and food samples were tested. The present study provides a novel experimental strategy for the development of sensors based on a DNAzyme to rapidly detect P. aeruginosa in the field.

Detection of Vibrio vulnificus in Seafood With a DNAzyme-Based Biosensor.

Vibrio vulnificus is an important pathogenic bacterium that is often associated with seafood-borne illnesses. Therefore, to detect this pathogen in aquatic products, a DNAzyme-based fluorescent sensor was developed for the in vitro detection of V. vulnificus. After screening and mutation, a DNAzyme that we denominated "RFD-VV-M2" exhibited the highest activity, specificity, and sensitivity. The limit of detection was 2.2 × 103 CFU/ml, and results could be obtained within 5-10 min. Our findings suggested that the target of DNAzyme RFD-VV-M2 was a protein with a molecular weight between 50 and 100 kDa. The proposed biosensor exhibited an excellent capacity to detect marine products contaminated with V. vulnificus. Therefore, our study established a rapid, simple, sensitive, and highly specific detection method for V. vulnificus in aquatic products.

Rapid Detection of Helicobacter pylori by the Naked Eye Using DNA Aptamers.

Helicobacter pylori was first isolated from gastritis patients by Barry J. Marshall and J. Robin Warren in 1982, and more than 90% of duodenal ulcers and about 80% of gastric ulcers are caused by H. pylori infection. Most detection methods require sophisticated instruments and professional operators, making detection slow and expensive. Therefore, it is critical to develop a simple, fast, highly specific, and practical strategy for the detection of H. pylori. In this study, we used H. pylori as a target to select unique aptamers that can be used for the detection of H. pylori. In our study, we used random ssDNA as an initial library to screen nucleic acid aptamers for H. pylori. We used binding rate and the fluorescence intensity to identify candidate aptamers. One DNA aptamer, named HPA-2, was discovered through six rounds of positive selection and three rounds of negative selection, and it had the highest affinity constant of all aptamers tested (K d = 19.3 ± 3.2 nM). This aptamer could be used to detect H. pylori and showed no specificity for other bacteria. Moreover, we developed a new sensor to detect H. pylori with the naked eye for 5 min using illumination from a hand-held flashlight. Our study provides a framework for the development of other aptamer-based methods for the rapid detection of pathogenic bacteria.

Recognition of Helicobacter pylori by protein-targeting aptamers.

BACKGROUND: Helicobacter pylori (H pylori) is a disease-causing pathogen capable of surviving under acidic conditions of the human stomach. Almost half of the world's population is infected with H pylori, with gastric cancer being the most unsatisfactory prognosis. Although H pylori has been discovered 30 years ago, the effective treatment and elimination of H pylori continue to be problematic. MATERIALS AND METHODS: In our study, we screened nucleic acid aptamers using H pylori surface recombinant antigens as targets. Trypsin was used for separating aptamers that were bound to proteins. Following nine rounds of screening, we performed sequence similarity analyses to assess whether the aptamers can recognize the target protein. Two sequences with desirable recognition ability were selected for affinity detection. Aptamer Hp4 with the strongest binding ability to the H pylori surface recombinant antigen was chosen. After optimization of the binding conditions, we conducted specificity tests for Hp4 using Escherichia coli, Staphylococcus aureus, Vibrioanguillarum, and H pylori. RESULTS: The data indicated that the aptamer Hp4 had an equilibrium dissociation constant (Kd ) of 26.48 ± 5.72 nmol/L to the target protein. This aptamer was capable of exclusively detecting H pylori cells, without displaying any specificity for other bacteria. CONCLUSIONS: We obtained a high-affinity aptamer for H pylori, which is expected to serve as a new molecular probe for detection of H pylori.