A Loop-Mediated Isothermal Amplification Integrated G-Quadruplex Molecular Beacon (LAMP-GMB) Method for the Detection of Staphylococcus aureus in Food

摘要

Staphylococcus aureus is a common pathogen in foodstuffs, and an effective detection assay is therefore essential for ensuring food safety. In this study, we established a label-free loop-mediated isothermal amplification integrated G-quadruplex molecular beacon (LAMP-GMB) chemiluminescence method for S. aureus detection based on its nuc gene that encodes a thermostable nuclease. Target DNA was amplified using the LAMP technique; the result LAMP products were specially recognized by GMB detection probe, subsequently induced the release of G-rich sequence, and formed a complex spatial structure G-quadruplex. The structure can bind with hemin and catalyze the H2O2-mediated oxidation of luminol reagent to produce the chemiluminescence signal for chemiluminescent detection. Under the optimized conditions, the developed method showed a good linear relationship (R-2=0.9882) in the range of 10(1)-10(5)fgL(-1) and was able to detect as low as 10fgL(-1) genomic DNA from S. aureus. Using spiked milk samples, the method exhibited a linear relationship (R-2=0.9893) ranging from 5x10(1) to 5x10(7)CFUmL(-1) with the limit of detection of 50CFUmL(-1). Importantly, the detection assay did not require the preparation and use of labeled primers for the LAMP reaction, increasing the reliability and efficiency of the assay. Therefore, the developed LAMP-GMB method has the potential to be widely applied to S. aureus monitoring work in food field.