Studies of the Real-time Rapid Detection of Staphylococcus aureus by Isothermal Target and Probe Amplification Assay

Introduction: In the last decade, Polymerase Chain Reaction (PCR) and Real-time PCR detection methods have been proposed for the detection of foodborne pathogens to replace the time-consuming, culture-based traditional techniques. They are rapid, easy to handle, sensitive, specific and constitute very valuable tools for food safety testing. However, the PCR method still has some disadvantages such as electrophoresis etc. complex operation, requiring expensive heating cycle equipment, and so on. The iTPA technology is simple to operate. It is a novel DNA amplification and fluorescent detection method for a foodborne pathogen by amplifying the target gene and the signal probe simultaneously under isothermal condition with high, specificity and sensitivity.

Purpose: In this study, we designed a set of specific primers and a probe according to the conserved regions of nuclease (nuc) gene of Staphylococcus aureus and developed a highly sensitive and specific real-time iTPA method for detecting food samples.

Methods: All bacterial strains used in this study were maintained in TSB. Seventy-seven S. aureus strains and 51 non-S. aureus strains were used to evaluate inclusivity and exclusivity. Sterilized water was used for the negative control. For comparison, a set of PCR reactions was performed using the iTPA outer primers. Specificity tests were repeated 3 times and compared to the traditional method. Real-time iTPA testing in experimentally inoculated various food samples.

Results: The S. aureus. nuc-based real-time iTPA assay successfully detected S. aureus strains while showing negative results for non-S. aureus strains, indicating that the nuc-based real-time iTPA assay was specific for S. aureus. The detection limits of the real-time iTPA assay using serial in S. aureus strain were determined and the lowest number of cells detected was 10² CFU/ml. The four primers and one FRET probe we designed from five regions of S. aureus nuc gene coding sequence that are highly specific to S. aureus.

Significance: A novel and rapid real-time DNA detection method has been developed using the real-time isothermal fluorescent equipment which is a much cheaper and simple operation than those of Real-time PCR equipments. We have demonstrated that real-time iTPA can be used to detect S. aureus down to 10² CFU/ml within 1 hour. The lowest detection limit achieved in this study was a 10¹ CFU per 25 g of food samples. So, S. aureus detection kit and iTPA Real time PCR equipment (iGene-S) was completed by using this method.