UV-ozone treated glass fiber based lateral flow DNA extraction platform integrated with LAMP for rapid detection of pathogen bacteria in whole blood

Abstract

The need for a fast and reliable platform for the detection of pathogens in point-of-care (POC) applications is increasing day by day. Molecular detection of pathogens is made possible by a good DNA isolation method, which allows for efficient and pure extraction from the cell. To address this need, our work presents a new lateral flow platform based on UV ozone-treated glass fiber pads for high-throughput DNA isolation from very low volumes of bacterial samples. The platform we developed mainly consists of UV ozone-treated glass fiber, which serves as the essential component binding DNA, and a cellulose-based absorbent pad acting as a reservoir for washing buffers and undesired substances from bacterial cells. UV ozone treatment increased the DNA recovery efficiency of glass fiber by 2.5 times compared to untreated glass fiber. qPCR results confirmed the isolation of pure DNA from both gram-negative and gram-positive bacteria samples performed on the developed platform. For five different bacteria concentrations (10–109 cfu/mL), a good linearity between Cq values and bacteria concentrations was obtained. A minimum concentration of 10 cfu/mL could be detected for both bacterial samples of 10 µL. The developed DNA extraction platform was also integrated with a portable heater for the LAMP-based detection application. We successfully demonstrated that the proposed extraction platform, combined with the colorimetric LAMP-based system, can detect Staphylococcus aureus bacteria spiked in whole blood samples in under one hour, including both extraction and detection processes. Our simplified paper-based platform will be a good alternative for the DNA extraction and molecular detection of pathogens without the need for complex laboratory equipment in POC applications.