Isothermal Target and Probe Amplification Assay for the Real-Time Rapid Detection of Staphylococcus aureus

Abstract

Staphylococcus aureus, the species most commonly associated with staphylococcal food poisoning, is one of the most prevalent causes of foodborne disease in Korea and other parts of the world, with much damage inflicted to the health of individuals and economic losses estimated at $120 million. To reduce food poisoning outbreaks by implementing prevention methods, rapid detection of S. aureus in foods is essential. Various types of detection methods for S. aureus are available. Although each method has advantages and disadvantages, high levels of sensitivity and specificity are key aspects of a robust detection method. Here, we describe a novel real-time isothermal target and probe amplification (iTPA) method that allows the rapid and simultaneous amplification of target DNA (the S. aureus nuc gene) and a fluorescence resonance energy transfer-based signal probe under isothermal conditions at 61 °C or detection of S. aureus in real time. The assay was able to specifically detect all 91 S. aureus strains tested without nonspecific detection of 51 non-S. aureus strains. The real-time iTPA assay detected S. aureus at an initial level of 10(1) CFU in overnight cultures of preenriched food samples (kiwi dressing, soybean milk, and custard cream). The advantage of this detection system is that it does not require a thermal cycler, reducing the cost of the real-time PCR and its footprint. Combined with a miniaturized fluorescence detector, this system can be developed into a simplified quantitative hand-held real-time device, which is often required. The iTPA assay was highly reliable and therefore may be used as a rapid and sensitive means of identifying S. aureus in foods.

Research advance in rapid detection of foodborne Staphylococcus aureus

“…Shin et al described a real-time isothermal target and probe amplification (iTPA) method that allowed the rapid and simultaneous amplification of the S. aureus nuc gene and a fluorescence resonance energy transferbased signal probe under 61 C isothermal conditions or detection of S. aureus in real time. [41] The results showed that the assay was able to specifically detect all 91 S. aureus strains tested without nonspecific detection of 51 nonÀS. aureus strains, and detect S. aureus at an initial level of 10 1 CFU in overnight cultures of preenriched food samples.…”

“…Copy number of the sample can be measured, rapid, high specificity and high sensitivity [28] Loop-mediated isothermal amplification 60 min 100 fg DNA/tube and 10(4) CFU/ml Specificity, simple operation, no special equipment, costless [34] Gene chip detection 60 min 10 1 CFU in overnight cultures of preenriched food samples Required only a water bath and a fluorometer and has great potential for use as a hand-held device [41] Biosensor technology 30 min 1000 cells/ml in pure culture Fast detection, with high cost [44]…”