Direct triplex loop-mediated isothermal amplification assay for the point-of-care molecular detection of Salmonella genus, subspecies I, and serovar Typhimurium

Highlights

• A triplex LAMP was developed to detect three genes in Salmonella simultaneously.

• A direct extraction method was optimized to improve the efficiency of LAMP reaction.

• Direct triplex LAMP could detect Salmonella at 6.4 × 10¹ CFU/g in chicken meat.

• This assay takes 60 min sampling-to-result to detect three target genes.

• This novel methodology could be utilized for point-of-care testing in various industry.

Abstract

In this study, a novel triplex loop-mediated isothermal amplification (LAMP) assay was developed and applied to a model of representative foodborne pathogen, Salmonella, to investigate the performance of the assay with high rapidity, specificity and sensitivity, and its ability to detect a pathogen in contaminated food. Three sets of LAMP primers were designed for target genes in Salmonella genus, subspecies I, and S. Typhimurium. Multiple targets were differentiated by the simultaneous analysis of annealing curves in a single LAMP reaction. In a triplex LAMP assay, each primer amplifies the target genes only, without any cross-reactivity with non-target genes. The limit of detection of this assay was 2.5 pg of S. Typhimurium DNA. The assay was applied to chicken meat artificially inoculated with S. Typhimurium. To decrease the inhibitory effects of the food matrix on the LAMP reaction, proteinase K was used in sample preparation, and the sample was heated and used directly as a DNA template. A combination of the direct extraction method optimized for food with triplex LAMP was able to detect low levels of S. Typhimurium (6.4 × 10¹ CFU/g) without a pre-enrichment step. This novel triplex multiplex LAMP assay successfully identified Salmonella genus, subspecies I, and S. Typhimurium within 60 min from sampling to results. This is therefore a rapid, reliable, sensitive, specific, and easy molecular diagnostic tool for on-site pathogen detection and food safety testing.

Introduction

Salmonella infection is recognized as a major health issue, and is associated with foodborne disease outbreaks worldwide. Salmonella is taxonomically divided into Salmonella enterica and S. bongori. According to studies conducted in numerous countries, S. enterica accounts for approximately 50% of all foodborne disease outbreaks (Furukawa et al., 2017; Jenkins, Dong, Li, & Higashiguchi, 2011). This species comprises six subspecies (subsp.): enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV), and indica (VI) (Buehler, Wiedmann, Kassaify, & Cheng, 2019). Among these, S. enterica subsp. enterica (I), accounts for 99% of all human Salmonella infections (Furukawa et al., 2017). Therefore, the identification of Salmonella at genus, subspecies, and serovar levels is important. There are over 1500 serovars in the subspecies I. Of these, S. Typhimurium is one of the Salmonella serovars most frequently associated with foodborne disease outbreaks (El-Aziz, 2013; Garrido-Maestu, Fucinos, Azinheiro, Carvalho, & Pardo, 2017; Park & Ricke, 2015).

To date, culture-based methods have primarily been used to screen for Salmonella in foods. These methods are reliable, but are time consuming and labor intensive (Azinheiro, Carvalho, Prado, & Garrido-Maestu, 2018; Park & Ricke, 2015; Wang et al., 2015). To address these problems, molecular technologies such as PCR and quantitative PCR (qPCR) have been used for the identification of Salmonella (Buehler et al., 2019; Vinayaka et al., 2019). In recent years, loop-mediated isothermal amplification (LAMP) has been used as a molecular diagnostic tool for the detection of pathogens. It is known to be more specific and sensitive than PCR or qPCR, because it can amplify six to eight specific regions of a target gene, unlike PCR or qPCR, which can target only two to three regions (Hu et al., 2018; Wang et al., 2015). The use of LAMP to amplify a target gene at a constant temperature simplifies the design and operation of the detection system, and allows the reaction to be completed more quickly than when using PCR-based methods (Zhang, Xu, Fohlerova, Chang, Iliescu, & Neusil, 2019). To obtain rapid and efficient detection, a multiplexing technique for the simultaneous detection of several targets in a single reaction can be combined with these assays. Recently, several studies using duplex LAMP for pathogen detection, food safety testing, and other applications have been conducted (Kim & Kim, 2018; Kubata & Jenkins, 2015; Liu et al., 2017; Wang et al., 2015). However, to the best of our knowledge, no report has simultaneously detected three genes of Salmonella in a single reaction using LAMP.

The aim of this study was to develop a triplex LAMP method for the simultaneous detection of Salmonella genus, Salmonella subsp. I and S. Typhimurium. STM3098 and STM4057 were selected as target genes for this assay. These genes have universally conserved sequences that can cover the entire Salmonella genus and Salmonella subsp. I, respectively (Kim, Park, & Kim, 2006; Park et al., 2009), as well as STMT4497, which is unique to S. Typhimurium, and can thus distinguish it from other S. enterica serovars (Kim, Park, Lee, et al., 2006). The triplex LAMP developed in this research was combined with a direct extraction method that was optimized in this study, and the applicability of this novel direct triplex LAMP assay was assessed using real food samples (Fig. 1).