Real-Time Loop-Mediated Isothermal Amplification (LAMP) Method for Quantitative Salmonella Typhi Detection Based on ViaB Gene

Abstract

Background & Objective: There is a vast range of diseases caused by Salmonella including gastroenteritis, enteric fever, bacteraemia, and focal infection. Salmonella infection is usually transmitted to humans by consuming foods or water contaminated with animal or human waste. This study aims to design a quantitative-LAMP method for real-time detection of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of Typhoid fever. Materials & Methods: A new LAMP primer set was designed and used based on the ViaB gene to specifically detect S. Typhi. The genome of some Salmonella-related and non- Salmonella-related bacteria was subjected to the LAMP assay for detection of S. Typhi to evaluate its analytical specificity, its analytical sensitivity and limit of detection (LOD). Further, turbidity in tubes was measured by a turbidimeter, and then a standard curve was depicted by plotting (Tt) time threshold values against the logarithm of ViaB gene copy number and this standard curve helped the researchers a lot to obtain a quantitative method. Results: The Salmonella Typhi LAMP assay specifically assessed the ViaB gene. It was shown that the analytical sensitivity of the assay was 0.28 fg using agarose gel electrophoresis or amplification plots in Loop amp real-time turbidimeter system, whereas the amplification was 2.8 fg under direct observation of fluorescent color changes. In the proposed methods, the lower limit of detection (LLOQ) of the assays was ~1 and 8 copies of the ViaB gene, respectively. In the LAMP assay for the quantitative detection of Salmonella Typhi, the linear correlation (r2= 0.97) was calculated between the log copy number and Tt values. Conclusions: The Salmonella Typhi LAMP assay is a simple and accurate tool to detect the causative agent of Typhoid fever that may be designed for clinical laboratories.