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.
Highlights
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
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)
Three sets of primers corresponding to the nuc of S. aureus, the fimY of Salmonella, and the ipaH of Shigella were designed (Table 2)
Summary
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. Shigellosis has high morbidity and mortality, 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)
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