Integrated silica membrane-based nucleic acid purification, amplification, and visualization platform for low-cost, rapid detection of foodborne pathogens.

Abstract

Real-time fluorescence detection of nucleic acid exhibit excellent performance in analytical and diagnostic applications. However, the requirement of laboratory-based instrument and complex nucleic acid extraction greatly limits their application in point-of-care testing (POCT). Herein, a novel integrated silica membrane-based platform incorporating nucleic acid purification, amplification, and detection steps was developed. A universal and portable visualization platform was fabricated by incorporating denaturation bubble-mediated strand exchange amplification (SEA) reaction with silica membrane. The fluorescence signal of SYBR Green I with amplification products was visualized by the naked eye using a simple ultraviolet light on the silica membrane, and significant discrimination between the positive and negative samples could be easily and visually obtained. Besides, chitooligosaccharide-modified silica membrane allows the purification of nucleic acid in a totally aqueous system and enables in situ SEA. With the proposed integrated platform, 102-108cfu/mL Vibrio parahaemolyticus could be successfully detected and excellent performance was also revealed for gram-positive pathogens. The detection limit of the method for artificially spiked oysters was 103cfu/g and reached 100cfu/g after 12h enrichment. This proof-of-concept method could also be applied to a variety of nucleic acid amplification methods. We believe that the proposed silica membrane-based platform has great potential for the rapid and low-cost detection of nucleic acids especially in low-resource settings. Graphical abstract.