Development of multiplex loop-mediated isothermal amplification for three foodborne pathogens

Abstract

Staphylococcus aureus, Salmonella, and Shigella are three major foodborne pathogenic microorganisms that cause global public health problems. We developed a multiplex loop-mediated isothermal amplification (mLAMP) assay for simultaneous detection of S. aureus nuc, Salmonella fimY, and Shigella ipaH in fresh fruit juice using three sets of primers. In addition, three different restriction enzyme cleavage sites were designed in each forward inner primer (FIP), namely, XhoI in nuc FIP, KpnI in fimY FIP, and BamHI in ipaH FIP. DNA was amplified using the LAMP assay at 64 °C for 50 min followed by endonuclease restriction digestion to separate the LAMP products of three pathogens. The minimum amount of genomic DNA of S. aureus, Salmonella, and Shigella that could be detected by mLAMP was 100 fg/25 μL, whereas for mPCR, it was 1 pg/25 μL. The artificially contaminated juice can also be detected by mLAMP after enrichment, which had the limit of detection (LOD) of 2 CFU/10 mL. In conclusion, the mLAMP developed in this study could be potentially used in the detection of S. aureus, Salmonella, and Shigella in food, particularly as a primary screening method in developing areas.

Keywords:
multiplex LAMP; Staphylococcus aureus; Salmonella; Shigella

1 Introduction

According to the World Health Organization (2015), unsafe food causes over 200 human diseases due to harmful bacteria, viruses, parasites or chemical substances, ranging from diarrhoea to cancers, and foodborne and waterborne diseases kill about two million people each year. Foodborne pathogens can cause a wide range of diseases including severe diarrhea and severe infections. Staphylococcus aureus, Salmonella enterica, and Shigella are three major foodborne pathogenic microorganisms that cause public health problems around the world. It is estimated that non-typhoidal S. enterica causes 93.8 million cases of acute gastroenteritis and 155,000 deaths every year around the world, of which 85% are foodborne (Food and Agriculture Organization of the United Nations, 2016). Shigellosis has high morbidity and mortality, particularly in resource-poor countries, where 167 million cases of diarrhea and over a million deaths occur annually (Von Seidlein et al., 2006). These three pathogens are responsible for 26.71% of bacteria foodborne events in China (Liu et al., 2016).

Based on the public health issue and economic losses caused by S. aureus, Salmonella and Shigella, these three pathogens should be monitored in foods (World Health Organization, 2003, 2015). The gold standard for S. aureus, Salmonella and Shigella testing is culture-based assays, which typically involve culture, identification, biochemical and serological detection. However, these assays are relatively complicated and time-consuming. Therefore, more rapid and simple methods are warranted, mainly includes immunology and molecular biology (Amani et al., 2015; Chen et al., 2015; Maerle et al., 2014; Nouri et al., 2018; Shao et al., 2011).

Loop-mediated isothermal amplification (LAMP) is a simple, rapid, sensitive, and specific method for the detection of pathogens that was developed by Notomi et al. (2000). Since it was developed, LAMP was used for detection and identification of viruses, bacteria and parasites (Huy et al., 2012; Imai et al., 2006; Shao et al., 2011; Sun et al., 2017). Compared to other detection methods, one of the most significant advantages of LAMP is its low cost, as this implies that it has a huge potential application value for pathogen detection. The purpose of this study was to develop an efficient and inexpensive assay for the detection of S. aureus, Salmonella, and Shigella, and assessed the application potential of mLAMP in food. This is the first time that mLAMP has been utilized in the simultaneous detection of S. aureus, Salmonella, and Shigella in juice using a single reaction.

2 Materials and methods

2.1 Bacterial strains and culture conditions

Seventeen standard strains were used for specificity and sensitivity testing in this study (Table 1). The culture medium of S. aureus, Salmonella, Shigella, and Escherichia coli was nutrient agar (NA), whereas Listeria monocytogenes was cultured in brain heart infusion (BHI). All strains were cultured at 37 °C.

Table 1
The 17 bacterial strains used in this study.

No.	Bacterial species	Strain serial number	Origin
1	Staphylococcus aureus	10384	China Center of Industrial Culture Collection, CICC
2	S. aureus	21600
3	S. aureus	21601
4	S. aureus	23656
5	Salmonella	21482
6	Salmonella	21484
7	Salmonella	21493
8	Salmonella	21513
9	Shigella	21534
10	Shigella	21535
11	Shigella	21680
12	Escherichia coli	10389
13	E. coli	10667
14	E. coli	21530
15	L. monocytogenes	21633
16	L. monocytogenes	21635
17	L. monocytogenes	23929

2.2 DNA extraction

Genomic DNA was extracted from each bacterial strain after overnight growth using a QIAGEN DNA extraction kit (DNeasy kit, QIAGEN, German). The concentration of the extracted DNA was determined at A260/280 by spectrophotometer (Shimadzu, UV-1700) and stored under at -20 °C.

2.3 Primer design

The sequences of the S. aureus nuc gene, S. enterica fimY gene, and Shigella ipaH gene were downloaded from NCBI and used in designing primers using Primer Explorer 5. Primer sequences were verified with BLAST® after design. Three sets of primers were used in the LAMP assays, and each set was used to amplify 17 DNA sequences of S. aureus, Salmonella, and Shigella (Table 1). XhoI was introduced into nuc FIP (Forward Inner Primer), KpnI was introduced into fimY FIP and BamHI was introduced into ipaH FIP (Table 2). Three sets of primers were used in this study (Table 2).

Table 2
Primers used in this study.

Primer name		Sequence (5'→3')
LAMP primers
nuc F3	AAAAGATGGTAGAAAATGCHAAG
nuc B3	TGTTCATGTGTATTGTTAGGTT
nuc FIP	ACGCTAAGCCACGTCCATATTCTCGAG(XhoI)AAAATTGAAGTCGAGTTTGACA
nuc BIP	TATGCTGATGGAAAAATGGTAAACGTAAACATAAGCAACTTTAGCCAAG
fimY F3	AGAAAGCTTTGCCTGTGG
fimY B3	WAACCTCGCTTATCGGAA
fimY FIP	AGCAAAGCGTACCTTATCATCGGGTACC(KpnI)GTTAAGGAGGGTGATAAGTTG
fimY BIP	GACGTGCTATTTCTTTTAAAGAGGCAGCTTTAGCCGTACTGAC
ipaH F3	GCTGGAAAAACTCAGTGCCT
ipaH B3	GGAACATTTCCCTGCCCA
ipaH FIP	CGACACGGTCCTCACAGCTCGGATCC(BamHI)TTCGACAGCAGTCTTTCGC
ipaH BIP	ATCTCCGGAAAACCCTCCTGGTAGCGCCGGTATCATTATCGA
PCR primers
nuc F3	AAAAGATGGTAGAAAATGCHAAG
nuc B3	TGTTCATGTGTATTGTTAGGTT
fimY F1	AAGGAGGGTGATAAGTTGTTT
fimY B1	AGCCGTACTGACTGGTTGA
ipaH F1	CGCGCTCACATGGAACAA
ipaH B1	AGTTTCTCTGCGAGCATGG

2.4 LAMP assay

Each LAMP reaction contained the following: 12.5 µL of a 2× Master Mix (WarmStart LAMP Kit, NEB, USA), 5 pmol each of F3 and B3, 40 pmol each of FIP and BIP (backward inner primer), 1 µL of DNA, and H₂O to a final reaction volume of 25 µL. LAMP reactions were performed under the isothermal condition of 64 °C for 50 min and then 80 °C for 5 min to terminate the reaction.

For multiplex LAMP (mLAMP), the following reaction componets were employed: 12.5 µL of a 2× Master Mix, 1.67 pmol each of nuc, fimY, and ipaH F3 and B3, 13.3 pmol each of nuc, fimY, and ipaH FIP and BIP, 1 µL each of DNA, and H₂O to a final reaction volume of 25 µL. Each assay was repeated three times.

2.5 mPCR assay

Multiplex PCR (mPCR) was conducted in parallel with the mLAMP assay. Each mPCR consisted of the following: 12.5 µL of a Taq premix (Premix Taq, TakaRa, China), 1.67 pmol of each nuc, fimY, and ipaH PCR primers, 1 µL each of S. aureus, Salmonella, Shigella DNA template, and H₂O to a final reaction volume of 25 µL. mPCR was performed using the following conditions: 94 °C for 3 min; followed by 30 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 20 s; and a final extension at 72 °C for 5 min. Each assay was repeated three times.

2.6 Restriction endonuclease analysis

The LAMP products were digested by restriction endonucleases XhoI, KpnI, and/or BamHI at 37 °C for 1 h. Each 30-µL restriction endonuclease system consisted of 3 µL of the restriction enzyme buffer, 1 µL of each enzyme, 3 µL of the LAMP product, and H₂O.

2.7 Specificity and sensitivity testing

The specificity of the LAMP assays used in this study was tested on 17 strains (Table 1), which included five foodborne pathogens. The specificity of the restriction endonuclease reactions was assessed by cross enzyme digestion. The LAMP products of S. aureus, Salmonella, and Shigella were digested by XhoI, KpnI, and BamHI separately.

To evaluate the sensitivity of the mLAMP assays, serial 10-fold dilutions of the DNA templates were used.

2.8 mLAMP for the simultaneous detection of S. aureus, Salmonella, and Shigella in artificially contaminated fresh fruit juice

The present study assessed the application potential of mLAMP in food. Artificially contaminated fresh fruit juices were prepared as described by Shao et al. and Garrido with modifications (Garrido-Maestu et al., 2017; Shao et al., 2011). The juices used in this study were purchased from a local supermarket. S. aureus, S. enterica, and Shigella were incubated in nutrient broth (NB) at 37 °C for shaking overnight. The concentration of three pathogens were determinated by CFU (colony forming unit) counting, meanwhile OD₆₀₀ was measured. Bacterium was diluted in NB, final inoculum concentration was adjusted to 100 CFU/mL, 50 CFU/mL, 10 CFU/mL, 5 CFU/mL, 2 CFU/mL, 1 CFU/mL. 3 mL of bacteria (1 mL of each) were added to 10 mL of juice, and mixed with 87 mL of NB. This matrix was incubated for 18 h at 37 °C. After incubation, 1 mL of matrix was taken and DNA was extracted following the DNA extraction above. Each inoculum concentration was repeated three times.

2.9 Product testing

The LAMP, PCR, and digestion products were analyzed by electrophoresis on a 2% agarose gel containing 0.5 μg/mL Andy Safe (Applied Bioprobes, USA).

3 Results

3.1 The specificity of LAMP assays and restriction endonuclease digestions

Multiplex LAMP was used to simultaneously detect S. aureus, Salmonella, and Shigella. Three sets of primers corresponding to the nuc of S. aureus, the fimY of Salmonella, and the ipaH of Shigella were designed (Table 2). Figure 1 shows that all three sets of primers were specific to their target templates, and no amplified DNA bands were detected after gel electrophoresis with non-target DNA (Figure 1).

Figure 1
Specificity of LAMP assays. Lanes: M, 2,000-bp DNA marker; 12~14, 3 Escherichia coli strains; 15~17, 3 Listeria monocytogenes strains; N, negative control; (a) 1~4, 4 Staphylococcus aureus strains; 5~8, 4 Salmonella strains; 9~11 3 Shigella strains; (b) Lanes: 1~4, 4 Salmonella strains; 5~8, 4 Staphylococcus aureus strains; 9~11 3 Shigella strains; (c) Lane: 1~3, 3 Shigella strains; 4~7, 4 Staphylococcus aureus strains; 8~11, 4 Salmonella strains. Source: by Cong XU.

In this study, S. aureus, Salmonella, and Shigella were detected by single-tube mLAMP assay, and restriction endonuclease digestion was used to analyze the mLAMP products due to the different enzyme cut sites which introduced in three FIPs. Specific enzyme digestions were important in discriminating different templates. Figure 2 shows that the LAMP products of S. aureus, S. enterica, and Shigella were only digested by XhoI, KpnI, and BamHI, respectively, as expected (Figure 2).

Figure 2
Specificity of restriction endonuclease reactions. Lanes: M, 2,000-bpDNA marker; 1,7,13, LAMP products of Staphylococcus aureus; 3, 9, and 15, LAMP products of Salmonella; 5, 11, and 17, LAMP product of Shigella; 2, 4, and 6, LAMP products of S. aureus, Salmonella, and Shigella digested by XhoI; 8, 10, and 12, LAMP products of S. aureus, Salmonella, and Shigella digested by KpnI; 6, 12, and 18, LAMP products of S. aureus, Salmonella, and Shigella digested by BamHI. Source: by Cong XU.

3.2 mLAMP assay and multiplex restrict endonuclease digestion

Three sets of primers and S. aureus, Salmonella, and Shigella DNA templates were added into one tube, where DNA was amplified; then the mLAMP products were analyzed by enzyme digestion. The DNA bands showed that the majority of the mLAMP products were digested by the three enzymes, whereas only a part of products were digested by XhoI, KpnI, or BamHI (Figure 3), meaning that there were three kinds of products in the mLAMP products and indicating that S. aureus, S. enterica, and Shigella DNA were all amplified in a single-vessel mLAMP system.

Figure 3
mLAMP assay and restriction endonuclease digestion. Lanes: M, 2,000-bp DNA marker; 1, mLAMP products of Staphylococcus aureus, Salmonella, and Shigella; 2~4, mLAMP products of S. aureus, Salmonella, and Shigella were separately digested by XhoI, KpnI, and BamHI separately; 5, mLAMP products of S. aureus, Salmonella, and Shigella were digested by XhoI, KpnI, and BamHI. Source: by Cong XU.

3.3 The sensitivity of mLAMP and mPCR assays

In this study, the sensitivity of the mLAMP assay was compared to the mPCR assay. Three pairs of primers were used in mPCR. Three pairs of primers were used in mPCR, namely, nuc F3/B3, fimY F1/B1, and ipaH F1/B1 were listed in Table 2. Figure 3 shows that a 10-fold dilution of DNA templates ranging from 1 ng to 1 fg per 25 μL was used to test the sensitivity of the mLAMP and mPCR assays. The DNA ladder patterns using 1 ng to 100 fg of DNA were clearly visible, indicating that the limit of detection (LOD) of mLAMP was 100 fg/25 μL, whereas that of mPCR was 1 pg/25 μL for target fragments of 181 bp, 126 bp, and 216 bp, as shown in the 1 ng to 1 pg lanes (Figure 4). Therefore, the mLAMP assay was 10 times more sensitive than the mPCR assay.

Figure 4
Sensitivity of mLAMP and mPCR assays. Upper panel: mPCR assay; lower panel: mLAMP assay. Lanes: M, 2,000-bp DNA marker; 1~7, Staphylococcus aureus, Salmonella, and Shigella DNA content was 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, 10 fg, and 1 fg; N, negative control. Source: by Cong XU.

3.4 mLAMP assay for artificially contaminated juice

To assess the applicability of mLAMP in practical testing, S. aureus, Salmonella, and Shigella were simultaneously inoculated into juice. S. aureus, Salmonella, and Shigella were detected by mLAMP when the initial concentration was >2 CFU/10 mL in the contaminated juice (Figure 5a). Furthermore, mLAMP products were digested by XhoI, KpnI, and BamHI, respectively, to distinguish the products of the three pathogens (Figure 5b).

Figure 5
Sensitivity of mLAMP assays for artificially contaminated juice. Lanes: M, 2,000-bp DNA marker; (a) 1~6, the numbers of S. aureus, Salmonella, and Shigella in artificially contaminated juice sample were 100, 10, 8, 5, 2, and 1 CFU/mL; N, negative control; (b) 1~3, mLAMP products of S. aureus, Salmonella, and Shigella were separately digested by XhoI, KpnI, and BamHI separately; 4, mLAMP products of S. aureus, Salmonella, and Shigella were digested by XhoI, KpnI, and BamHI. Source: by Cong XU.

4 Discussion

S. aureus, Salmonella, and Shigella are three important foodborne pathogens that are largely responsible for foodborne illnesses occurring around the world (Wang et al., 2007; World Health Organization, 2015), particularly in developing countries (Von Seidlein et al., 2006). An effective and rapid monitoring mean is thus essential for the control of these three bacterial infections.

LAMP has become a powerful tool to detect foodborne pathogens since it was developed (Maerle et al., 2014; Wang et al., 2007, 2008). Yang et al. (2011) previously reported that the sensitivity of LAMP in detecting S. aureus was 1.25 CFU per reaction tube and 10.3 CFU per reaction in the artificial contamination test. Garrido-Maestu et al. (2017) reported systematic loop-mediated isothermal amplification assays were able to detect <10 cfu/25 g food samples. Shao et al. (2011) established mLAMP to simultaneously detect Salmonella spp. and Shigella spp. in milk, and their detection limits were 100 fg DNA/ tube with genomic DNA and initial inoculation levels of 5 CFU/10 mL. Wang et al. (2015) reported a multiple endonuclease restriction real-time LAMP technology for simultaneously differentiating Shigella spp. and Salmonella spp. using 62.5 and 125 fg DNA per tube, respectively.

In the present study, restriction enzyme cleavage sites were designed in each FIP primer, namely, XhoI in nuc FIP, KpnI in fimY FIP, and BamHI in ipaH FIP. After mLAMP, restriction digestion was performed to distinguish S. aureus, Salmonella, and Shigella or a DNA mixture of any of the three pathogenic bacteria. Both LMAP and restriction digestion were pathogen-specific. The mLAMP LOD for each pathogen were 100 fg DNA/25 μL which is identical to Wang et al. (2015), and 2 CFU/10 mL of initial inoculation in juice. The sensitivity of mLAMP was 10-fold higher than mPCR, which is the same as that of previous reports (Shao et al., 2011; Wang et al., 2015). Furthermore, in this study, mLAMP can be completed within 20 h (including culture, DNA extraction, LAMP, enzyme digestion and gel analysis) and is thus superior to traditional methods that are performed within the range of five to seven day (based on the methods protocols). In addition, mLAMP and endonuclease restriction only require a heating block, which is a common laboratory equipment. This study could be applied on preliminary detection of S. aureus, Salmonella, and Shigella when SYBR GREEN I is added in the reaction system (data not shown). For as long as any kinds of the above-mentioned bacteria exist in the sample, they could be detected by direct observation, with no need for electrophoresis and enzyme reaction in the preliminary estimation. Restriction enzyme digestion could be further applied in order to determine the species of the pathogenic bacterium.

In conclusion, this study provides a rapid, specific, sensitive, and low-cost assay for the simultaneous detection of S. aureus, Salmonella, and Shigella. The detection of these three bacterial pathogens in juice indicates that mLAMP could be potentially used in batches food screening in basic and field laboratories.

Acknowledgements

The Science and Technology Project of Guangdong Province (No. 2017A020208002) supported this study.

• Practical Application: Detection of foodborne pathogens.

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Publication Dates

• Publication in this collection
  28 Nov 2019
• Date of issue
  June 2020

History

• Received
  18 Mar 2019
• Accepted
  24 Aug 2019