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Evaluation of a Loop-Mediated Isothermal Amplification (LAMP) Method for the Detection of Salmonella spp. in Terms of Sensitivity and Applicability

Salmonella spp. are important food-borne pathogens that can cause diseases in humans. Many detection methods have been established in Salmonella spp. using loop-mediated isothermal amplification (LAMP) or reverse transcription loop-mediated isothermal amplification (RT-LAMP). The detection limits of these assays varied from 1 CFU/reaction to 104 CFU/reaction, from 100 fg genomic DNA/reaction to 10 pg genomic DNA/reaction, or from 2.0×101 CFU/mL to 107 CFU/mL for food samples. In this study, LAMP assays were developed using genomic DNA for the detection of Salmonella spp. Two sets of LAMP primers were designed using the invA gene and the 16S-23S rRNA intergenic spacer region (ITS) of S. enterica as the target sequences for two LAMP assays. The detection limits of the two methods were respectively 20 pg S. enterica DNA/reaction and 10 pg S. enterica DNA/reaction at the optimized temperature, and the LAMP methods were of high repeatability and specificity for S. enterica detection. This study provides a baseline for the application of LAMP for the detection of food-borne pathogenic bacteria. © 2020 Yanhong Liu. Hosting by Science Repository. All rights reserved.


Introduction
Food-borne diseases caused by pathogenic bacteria have become a major global public health issue, among which salmonellosis is the illness most often attributed to the consumption of poultry, beef, pork, eggs, milk, seafood, nut products, and fresh produce contaminated by Salmonella spp. [1,2]. Salmonella spp. have been identified as the most frequent cause of food-borne infection outbreaks in many countries [3]. Therefore, rapid and sensitive methods for the detection of Salmonella spp. are needed to control and prevent salmonellosis outbreaks. Many methods, including culture-based, immunology-based, and nucleic acidbased, have been developed, among which more than twenty loopmediated isothermal amplification (LAMP) or reverse transcription loop-mediated isothermal amplification (RT-LAMP) methods have been established targeting different specific genes of Salmonella spp. [3].
Most of these LAMP methods used the invasion gene invA as the target sequence [4][5][6][7][8][9][10][11][12][13]. For example, Chen et al. developed specific LAMP methods utilizing the invE gene and 3 serotype-specific genes, fliC, lygD and STM4495 for the detection of Salmonella and 3 common Salmonella serotypes [14]. In the study of Tang et al., the LAMP assay with the fimY gene as the target sequence was established to detect Salmonella species in possibly infected ducks [15]. Okamura et al. developed the LAMP assays that amplified the rfbJ gene for detection of the O4 group of S. enterica and the insertion element of SE for detection of the O9 group of Salmonella in chickens [16,17]. A sefA-based LAMP method was developed by Gong et al. for detection of S. enteritidis and S. gallinarum in chickens [18]. The phoP, hisJ, and rfbS genes of Salmonella spp. have also been used as target sequences for LAMP assays [2]. In spite of different genes as target sequences, all of these LAMP or RT-LAMP assays were of high specificity for Salmonella spp., but they varied greatly in sensitivity (detection limit) When CFU/reaction, cfu/test, copies/reaction, and cells/test were used as the measurement units, the detection limits of 1 CFU per reaction, >2.2 cfu/test, 4 CFU/reaction, 6.0 cfu/test and 4.8 cfu/test, 15 copies/reaction, and 10 4 CFU/reaction, have been reported [4,9,12,15,18,19]. With CFU/mL, CFU/25 ml, CFU/g and CFU/25g as the measurement units for detection of Salmonella spp. in food produces, the detection limits were determined to be 2.0×10 1 CFU/mL, 10 2 CFU/25g, 200 CFU/g, 10 3 CFU/ml, 10 4 CFU/25 ml, 10 4 -10 6 CFU per 25 g, and 10 7 CFU/mL [8, 11-13, 17, 19].
When pg DNA/tube, fg DNA/tube, and pg/μL were used as the measurement units, the sensitivity of the LAMP assays was found to be 100 fg DNA/tube, 1 pg DNA/tube, and 10 pg/μL [3,5,7]. In conclusion, since the results of these LAMP assays in detection limits have been confusing to interpret, the LAMP technology has not been practically applied for the detection of Salmonella spp. In this study, genomic DNA from pure bacterial cultures of Salmonella spp. was used as template, LAMP assays with the invA gene and 16S-23S rRNA intergenic spacer region of S. enterica as the target sequences were developed, the specificity and sensitivity of LAMP assays were also evaluated.

I Primer Design for LAMP Assay
Two sets of LAMP primers targeting the invA gene (GenBank accession No. CP041208.1) and the 16S-23S ribosomal RNA intergenic spacer (ITS) (GenBank accession No. CP050716.1) of Salmonella enterica serotype Newport were designed using PrimerExplorer 5 (Link), and one set of primers was selected with Oligo 7 (Molecular Biology Insights, Inc. Colorado Springs, USA). The primer sequences are listed in (Table  1). Table 1: LAMP primers targeting invA and ITS of Salmonella enterica.

II Bacteria Strains and Genomic DNA Extraction
The Salmonella enterica and non-Salmonella enterica strains used in this study are listed in ( Table 2). All of the strains except the Listeria monocytogenes strains were cultured overnight at 37°C in Luria-Bertani (LB) broth, and the L. monocytogenes strains were cultured in DifcoTM Buffered Listeria Enrichment Broth Base (Becton, Dickinson and Company). Genomic DNA from the pure cultures was extracted using the DNeasy® Blood & Tissue Kit (QIAGEN N.V. Corporate), and the DNA templates were used to determine the sensitivity and specificity of the LAMP assays.

III Sensitivity Determination
The LAMP assay using the invA gene primers was performed in a 25 μL reaction mixture containing 0.

IV Specificity Determination
The specificity of two LAMP assays with invA primers and ITS primers were determined using the bacterial strains listed in (Table 2), and the amount of DNA template used was 100 pg per reaction.

I Detection Limit of the LAMP Assays
As shown in (Figure 1), the detection limit of the LAMP assay with invA primers was 20 pg per reaction, while the sensitivity of the LAMP assay with 16S-23S ribosomal RNA intergenic spacer as target sequence was 10 pg per reaction, and there was little significant difference in terms of detection limits for these two LAMP assays.     (Table 2).

II Specificity of the LAMP Assay with ITS Primers
The specificities of the LAMP assay with ITS primers and the LAMP assays with invA primers were determined. As shown in (Figure 2), as for both LAMP assays, the six repeats of Salmonella enterica serotype Newport were positive, while the two repeats of 22 non-Salmonella enterica strains and two negative controls were negative; therefore, the LAMP assays were highly specific for Salmonella enterica.

Discussion
Two LAMP assays with the invA gene and 16S-23S rRNA intergenic spacer region of S. enterica as the target sequences were established in this study. The sensitivity of the assay with ITS primers (10 pg per reaction) was slightly higher than that of the assay with invA primers (20 pg per reaction), which is equivalent to 3×10 4 copies/reaction. The sensitivity was similar to that of the LAMP assays developed by Liu et al. and Zhang et al., but it was much lower than that of other reported LAMP assays in which there was up to four orders of magnitude difference [3-5, 9, 12, 14, 15, 18, 19].
One important factor for differences in sensitivity is that the DNA preparation methods can have a great effect on the LAMP reaction. When the DNA template was prepared by a boiling method, the detection limit was as low as 1 CFU per reaction; it was proposed that the boiling and cooling process provided the single-strand template for the LAMP reaction [4,5,10,[12][13][14][15]. A commercial DNA preparation kit was used by Liu et al., and in our study, the prepared DNA template was double stranded, and the sensitivity was as low as 10 pg per reaction [3]. The mechanisms of Polymerase Chain Reaction (PCR), Helicase-dependent Amplification (HDA) and Recombinase Polymerase Amplification (RPA) assays for amplification of double stranded DNA are wellestablished; however, there is so far no report on the mechanism of the LAMP reaction with double stranded DNA as template, which warrants further study.
Other factors related to sensitivity include the method used to determine results, and use of enrichment, reverse transcription, etc. It was reported by Zhang et al. that the reduction of the detection limit, from 10 4 copies/reaction to 10 2 copies/reaction, could be the result of using visual determination by fluorescence instead of turbidity [9]. The LAMP assays could detect 10 4 CFU/25 ml without enrichment and 100 CFU/25 ml with an 8 h enrichment for Salmonella spp., Techathuvanan et al. improved the Salmonella detection limit from 10 6 CFU/25 g to 10 2 CFU/25 g for both pork chop and sausage samples using a 10 h enrichment [8,12]. With detection of Salmonella spp. with RT-LAMP, the 6 h enrichment made little difference and only improved the detection limits from 10 7 CFU/25 mL to 10 6 CFU/25 mL [11]. It could be that competitive or non-competitive inhibitors existed between reactions catalyzed by reverse transcriptase and Bst DNA polymerase (Large Fragment), which also needs further study.
The LAMP assays with the invA gene and the 16S-23S ribosomal RNA intergenic spacer as the target sequence were established in this study. Like most previous reports, they were highly specific to Salmonella enterica [2]. It was also proven by Wang et al. that the presence of high concentrations of non-target genomic DNA neither adversely affected the amplification efficiency nor generated significant background [5]. In conclusion, the LAMP assay can be used to detect Salmonella spp. in different food matrices sensitively and selectively, given that a proper DNA preparation method was used after enrichment.