SOMAprobes, Science and Technology Park of Gipuzkoa, Donostia-San Sebastian, 20009, Spain.
Animal Health Research Group, Institute of Agrobiotechnology, CSIC-UPNa-Gobierno de Navarra, Mutilva, 31192, Spain.
Anal Chim Acta. 2019 Apr 25;1054:157-166. doi: 10.1016/j.aca.2018.12.027. Epub 2018 Dec 21.
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 10 CFU mL for STM 1 and 10 CFU mL for SE 1, while Sal-5 proves to be a slightly better probe, with LODs of 10 CFU mL for STM 1 and 10 CFU mL 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.
沙门氏菌是人类细菌性食源性疾病的主要源头，可引发肠胃炎暴发，并伴有菌血症，从而导致临床并发症和死亡。鸡蛋、家禽和猪肉产品被认为是人类感染致病性沙门氏菌的主要载体。为了预防这种相关的人畜共患病，食品安全法规进行了关键改革，以加强食品生产链的控制。尽管采取了这些措施，但在过去十年中，全世界仍报告了大规模的沙门氏菌病疫情。因此，开发沙门氏菌检测的新策略是食品安全和公共卫生当局的当务之急。与目前可用的方法（数天）相比，此类检测系统应大大缩短诊断时间（数小时）。在此，我们报告了用于在孵育不到 8 小时内灵敏和特异性检测活沙门氏菌的核酸探针的发现和特性。我们是第一个假设源自沙门氏菌的核酸酶活性作为感染生物标志物，并将其用于开发创新检测策略的人。我们的研究结果表明，筛选并鉴定了两种寡核苷酸序列（底物）作为检测沙门氏菌的最有前途的探针，即 Sal-3 和 Sal-5。使用参考沙门氏菌 Typhimurium（STM 1）和沙门氏菌 Enteritidis（SE 1）培养物确定了这两种探针的检测限。Sal-3 对 STM 1 和 SE 1 的检测限约为 10 CFU mL，而 Sal-5 则被证明是一种稍好的探针，其对 STM 1 和 SE 1 的检测限均为 10 CFU mL。这两种选定的探针均已证明能够识别体外测试的 51 种不同沙门氏菌血清型中的 49 种，并且能够识别作为肥育猪沙门氏菌基线研究标准样品的猪肠系膜淋巴结中的最常见血清型。值得注意的是，我们的结果表明，核酸酶检测与 PCR-InvA 或 ISO-6579 标准方法之间具有 100%的相关性，这突显了这种创新的核酸技术作为食品安全检测快速方法的巨大潜力。
