The Development and Evaluation of a Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Salmonella enterica serovar Typhi

Abstract

Typhoid fever remains a public health threat in many countries. A positive result in traditional culture is a gold-standard for typhoid diagnosis, but this method is time consuming and not sensitive enough for detection of samples containing a low copy number of the target organism. The availability of the loop-mediated isothermal amplification (LAMP) assay, which offers high speed and simplicity in detection of specific targets, has vastly improved the diagnosis of numerous infectious diseases. However, little research efforts have been made on utilizing this approach for diagnosis of Salmonella enterica serovar Typhi by targeting a single and specific gene. In this study, a LAMP assay for rapid detection of S. Typhi based on a novel marker gene, termed STY2879-LAMP, was established and evaluated with real-time PCR (RT-PCR). The specificity tests showed that STY2879 could be amplified in all S. Typhi strains isolated in different years and regions in China, whereas no amplification was observable in non-typhoidal strains covering 34 Salmonella serotypes and other pathogens causing febrile illness. The detection limit of STY2879-LAMP for S. Typhi was 15 copies/reaction in reference plasmids, 200 CFU/g with simple heat-treatment of DNA extracted from simulated stool samples and 20 CFU/ml with DNA extracted from simulated blood samples, which was 10 fold more sensitive than the parallel RT-PCR control experiment. Furthermore, the sensitivity of STY2879-LAMP and RT-PCR combining the traditional culture enrichment method for simulated stool and blood spiked with lower S. Typhi count during the 10 h enrichment time was also determined. In comparison with LAMP, the positive reaction time for RT-PCR required additional 2-3 h enrichment time for either simulated stool or blood specimens. Therefore, STY2879-LAMP is of practical value in the clinical settings and has a good potential for application in developing regions due to its easy-to-use protocol.

Rapid and specific detection of Tilletia indica using loop-mediated isothermal DNA amplification

“…Some studies have found LAMP is able to detect five to a few tens of copies of plasmid and genomic DNA, a detection sensitivity that is 10 times higher than conventional PCR (Fan et al 2015;Nakao et al 2010;Notomi et al 2000;Zhou et al 2014). …”

Loop-mediated isothermal amplification (LAMP): a versatile technique for detection of micro-organisms

“…1 was modified from Notomi et al (2000). [Colour figure can be viewed at wileyonlinelibrary.com] instance, Salmonella typhi (Abdullah et al 2014;Fan et al 2015), Campylobacter jejuni and Campylobacter coli (Pham et al 2015), and Helicobacter pylori (Bakhtiari et al 2016;Yari et al 2016). Other successful examples of LAMP usage include Neisseria meningitidis (Lee et al 2015), Listeria monocytogenes (Birmpa et al 2015;Wang et al 2015b;Ye et al 2015), Entamoeba histolytica (Rivera and Ong 2013;Singh et al 2013;Mwendwa et al 2017), and human leptospirosis (Seesom et al 2015;Hsu et al 2017).…”

“…However, they still suffer from insufficiencies and inadequacies. All these methods require a sophisticated instrument for amplification and detection or elaborate methods for performance (Fang et al 2008). As a result, researchers and scientists have worked hard to overcome these limitations.…”

Development of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of <i>Cannabis sativa</i>

“…29) To simplify the DNA extraction procedure, a simple method for sample preparation was evaluated for the LAMP assay. 30,31) Supernatants of heat-treated samples following proteinase treatment were used as DNA template. As indicated in Fig.…”