Abstract
Staphylococcus aureus strains carrying enterotoxin A gene (sea) causes food poisoning and cannot be distinguished from non-pathogenic strains by the culture method. Here, we developed a rapid, specific and sensitive visual detection of sea using loop-mediated isothermal amplification (LAMP) combined with nanogold probe (AuNP) or styryl dye (STR). LAMP-AuNP and LAMP-STR can detect as low as 9.7 fg (3.2 sea copies) and 7.2 sea copies, respectively, which were lower than PCR (97 fg or 32 sea copies). The excellent performance of these new assays was demonstrated in food samples using crude DNA lysates. While the culture method detected 104 CFU/g in ground pork and 10 CFU/mL in milk in 5–7 days, LAMP-AuNP could detect down to 10 CFU/g for both samples in 27 minutes. Analyzing 80 pork and milk samples revealed that the LAMP-AuNP showed 100% sensitivity, 97–100% specificity and 97.5–100% accuracy, which were superior to the culture method, and comparable to PCR but without requirement of a thermal cycler. Furthermore, our LAMP-AuNP detect sea at a range below the food safety control (<100 CFU/g). The LAMP-STR quantitated sea in 10–1,000 CFU (7.2–720 copies). Our crude DNA lysis combined with LAMP-AuNP/STR present effective point-of-care detection and facilitate appropriate control strategies.
Highlights
Staphylococcus aureus strains with the staphylococcal enterotoxin A gene bacteriophage, are a common cause of foodborne disease and outbreaks worldwide including the United States and countries of the European Union[1,2,3,4,5]
Since the visual reading from loop-mediated isothermal amplification (LAMP)-AuNP cannot quantitate the number of bacterial colony forming unit (CFU), which is important when the contamination level is below the food and drink safety control (
Conditions were optimized over a temperature range of 60–65 °C and incubation periods of 15–60 min using 100 ng genome of S. aureus strains sea as template, and our designed LAMP primers that consisted of two inner loop primers (Supplemental Table 2, Sa_SEA LF and Sa_SEA LB) versus Goto et al.’s LAMP primers[2] that contain only one inner loop primer (SEA_LB)
Summary
Staphylococcus aureus strains with the staphylococcal enterotoxin A gene (sea) bacteriophage, are a common cause of foodborne disease and outbreaks worldwide including the United States and countries of the European Union[1,2,3,4,5]. An alternative method for highly specific analysis of a food-borne pathogen is the nucleic acid-based assay, which generally requires amplification prior to detection to achieve the desired sensitivity. The LAMP-AuNP assay was validated by testing commonly contaminated food samples (ground pork and milk) to show improved specificity and sensitivity[26,27,28] compared to the standard nucleic acid-based assay (PCR). Since the visual reading from LAMP-AuNP cannot quantitate the number of bacterial CFU (or copy), which is important when the contamination level is below the food and drink safety control (
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