Detection of Salmonella enterica subsp. enterica via Quenching of Unincorporated Amplification Signal Reporters in Loop-Mediated Isothermal Amplification

Abstract

Salmonella enterica is a major cause of diarrheal diseases in developing countries where timely surveillance and proper clinical management are inadequate. In this study, a rapid and cheap method of detecting S. enterica DNA was developed by employing the Quenching of Unincorporated Amplification Signal Reporters in Loop-Mediated Isothermal Amplification (QUASR LAMP) platform. QUASR LAMP provides a closed-tube, target-specific endpoint detection of pathogens, wherein results can be analyzed visually through an LED transilluminator and verified through agarose gel electrophoresis. Based on the chromosomal SopD gene, primers and probes were first designed, then screened. The assay was subsequently optimized so that the presence of S. enterica is determined by incubating the extracted DNA at 65°C for only 60 minutes. The assay was repeatable and can be performed by simply using a thermal cycler or even a dry bath incubator. S. enterica positives appear bright yellow green when viewed through a yellow filter excited with blue LED. The developed assay had an in silico and in vitro specificity towards Salmonella enterica subsp. enterica serovars with a limit of detection of 104 copies per microliter. The Salmonella QUASR LAMP assay has the potential for food and environmental applications. Chiefly, as an alternative to traditional microbiology and PCR, this QUASR LAMP assay can be used for point-of-care salmonellosis testing of clinical specimens in low-resource settings.