This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (CC BY).
METHOD
Detection of Salmonella enterica by loop-mediated isothermal amplification of DNA using a fluorescently labeled loop primer
1 Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia
2 Speransky City Children’s Clinical Hospital No. 9, Moscow, Russia
3 Institute of Translational Medicine and Biotechnology, Sechenov University, Moscow, Russia
Correspondence should be addressed: Mikhail L. Filipenko
Prospekt Lavrenteva, 8, Novosibirsk, Russia; moc.liamg@oknepiliflm
Funding: This work was supported by the Russian state-funded project for the Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences (Grant No. 125012300671-8).
Author contribution: Shamovskaya DV, Smertina MA, Khrapov EA — LAMP assay optimization and validation on clinical samples; Gordukova MA, Galeeva EV — collection of clinical specimens and patient data; Oskorbin IP — experimental design for LAMP assays; Boyarskikh UA — LAMP primer design; Filipenko ML — conceptualization, writing, and editing of the manuscript.
Compliance with ethical standards: The study was approved by the Ethics Committees of the Institute of Chemical Biology and Fundamental Medicine, SB RAS (Protocol No. 4, May 25, 2022) and the City Clinical Hospital for Children No. 9 named after G. N. Speransky, Moscow Healthcare Department (Protocol No. 44, April 19, 2022). All patients provided written informed consent.
Salmonellosis remains one of the leading causes of bacterial gastrointestinal infections in humans and animals. Molecular diagnostics has dramatically reshaped the diagnostic landscape for Salmonella infection; however, it remains time- and resource-intensive. Isothermal DNA amplification, for example loop isothermal amplification (LAMP), performed at a constant temperature, is the basis for the development of rapid diagnostic tests that can be adapted to the point-of-care (PoC) formats and implemented in resource-limited settings or remote from centralized laboratories. The aim of this study was to develop and validate a novel LAMP-based method for detecting Salmonella enterica in human stool samples, wherein amplification results are monitored using a loop primer labeled with a fluorophore and an internal quencher. The proposed method achieves a limit of detection (LoD95) of 250 copies per reaction, with a sensitivity of 86.84% (95% CI: 71.91–95.59%) and specificity of 96.49% (95% CI: 87.89–99.57%) relative to qPCR, and demonstrates increased robustness against DNA amplification inhibitors present in fecal samples. Incorporation of distinct fluorophores into loop primers for FLP-LAMP targeting different genes could potentially enable multiplexing and simultaneous detection of multiple pathogens, thereby expanding the diagnostic utility of isothermal amplification.
Keywords: fluorescent probe, LAMP, Salmonella enterica, fluorescent loop primer, analytical validation, DNA amplification inhibitors