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

Abstract

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 hours 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 10 5 CFU mL -1 for STM 1 and 10 4 CFU mL -1 for SE 1, while Sal-5 proves to be a slightly better probe, with LODs of 10 4 CFU mL -1 for STM 1 and 10 4 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.

Point-of-Need DNA Testing for Detection of Foodborne Pathogenic Bacteria

“…Successfully designed DNA probes has led to many biosensor developments for applications in food safety, including different Salmonella serotypes [67], Campylobacter spp. [68], L. monocytogenes [69] or hepatitis A virus [70].…”

Rational Design and Experimental Analysis of Short-Oligonucleotide Substrate Specificity for Targeting Bacterial Nucleases

“… 1 In particular, the use of nuclease-activatable oligonucleotide probes (NAOPs) as biosensors has proven its potential as a groundbreaking solution in early diagnosis of serious infectious diseases. 2 , 3 The detection of bacterial infection, 4 − 6 malignant cells in biopsies of breast cancer, 7 − 9 or food contamination by pathogenic bacteria 1 has been previously reported to be feasible on the basis of their nuclease activity profile. 10 − 12 A key aspect in the development of any diagnostic tool based on oligonucleotide probes is the necessity of designing probes which are both efficient and specific.…”

Comparison of Untreated, Lime-Stabilized and Composted Wastewater Sludges from a Pulp, Board and Paper Mill Integrate as a Fertilizer Product

“…The details of the analytical methods of the standards can be found in other publications [Pöykiö et al, 2019]. Temperatureresistant coliforms and Escherichia coli in the sludges were determined by the NMKL method 125 [Luukkonen et al, 2020] and Salmonella by the standard ISO 6579 [Machado et al, 2019].…”