Kinetic Screening of Nuclease Activity using Nucleic Acid Probes.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable. Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes' design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Rapid and specific detection of Salmonella infections using chemically modified nucleic acid probes.

Salmonella is a leading source of bacterial foodborne illness in humans, causing gastroenteritis outbreaks with bacteraemia occurrences that can lead to clinical complications and death. Eggs, poultry and pig products are considered as the main carriers of the pathogenic Salmonella for humans. To prevent this relevant zoonosis, key changes in food safety regulations were undertaken to improve controls in the food production chain. Despite these measures, large outbreaks of salmonellosis were reported worldwide in the last decade. Thus, new strategies for Salmonella detection are a priority for both, food safety and public health authorities. Such detection systems should provide significant reduction in diagnostic time (hours) compared to the currently available methods (days). Herein, we report on the discovery and characterization of nucleic acid probes for the sensitive and specific detection of live Salmonella within less than 8 h of incubation. We are the first to postulate the nuclease activity derived from Salmonella as biomarker of infection and its utility to develop innovative detection strategies. Our results have shown the screening and identification of two oligonucleotide sequences (substrates) as the most promising probes for detecting Salmonella - Sal-3 and Sal-5. The detection limits for both probes were determined with the reference Salmonella Typhimurium (STM 1) and Salmonella Enteritidis (SE 1) cultures. Sal-3 has reported LOD values around 105 CFU mL-1 for STM 1 and 104 CFU mL-1 for SE 1, while Sal-5 proves to be a slightly better probe, with LODs of 104 CFU mL-1 for STM 1 and 104 CFU mL-1 for SE 1. Both selected probes have shown the capability to recognize 49 out of 51 different Salmonella serotypes tested in vitro and the most frequent serotypes in porcine mesenteric lymph nodes as a standard sample used in fattening-pig salmonellosis baseline studies. Notably, our results showed 100% correlation between nuclease detection and the PCR-InvA or ISO-6579 standard method, underlining the great potential of this innovative nucleic acids technology to be implemented as a rapid method for food safety testing.