Optimization for Rapid Detection of Staphylococcus aureus using Real-time LAMP

Introduction: Bacterial food poisoning is considered a global concern in terms of economical and human health. Staphylococcus aureus produces low molecular weight extracellular toxins. These enterotoxins (SEs) are similar in structure and bioactivity. Today, there are several methods to detect SE genes. Nevertheless, culture-based and immunological methods are simple and cheaper than molecular techniques, they are time-consuming, with low sensitivity and specificity. In this field, loop-mediated isothermal amplification (LAMP) is a fast and simple method for gene amplification. The aim of this work was to optimize the LAMP reaction using the Taguchi method.Materials and Methods: For this, in order to improve and accelerate the LAMP diagnostic process, essential factors for the identification of enterotoxin, including MgSO۴ concentration, time, and temperature reaction were optimized separately and using Taguchi experimental design.Results: The results showed that ۵۷ µg/ml of S. aureus genome as template is suitable for the replication, and in optimization of LAMP assay in separate condition the best replication rate was observed at ۶ mM MgSO۴, ۴۵ min, and ۶۵ °C. Whereas using Taguchi methods, the optimum condition was at ۸ mM MgSO۴, ۶۰ min and ۶۵ °C.Conclusions: The one-step-visual developed LAMP assay with the optimum conditions could be of interest for screening functions in food analytical laboratories, and a portable detection method could be used to design a suitable identification kit for S. aureus without the need for special equipment or trained personnel.

کلیدواژه ها:

Food poisoning ، Staphylococcus Aureus ، Enterotoxin ، Detection ، optimization