Micromachines (Apr 2023)
A Lab-on-a-Tube Biosensor Combining Recombinase-Aided Amplification and CRISPR-Cas12a with Rotated Magnetic Extraction for <i>Salmonella</i> Detection
Abstract
Background: Foodborne pathogenic bacteria threaten worldwide public health, and simple bacterial detection methods are in urgent need. Here, we established a lab-on-a-tube biosensor for simple, rapid, sensitive, and specific detection of foodborne bacteria. Methods: A rotatable Halbach cylinder magnet and an iron wire netting with magnetic silica beads (MSBs) were used for simple and effective extraction and purification of DNA from the target bacteria, and recombinase-aided amplification (RAA) was combined with clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins12a(CRISPR-Cas12a) to amplify DNA and generate fluorescent signal. First, 15 mL of the bacterial sample was centrifuged, and the bacterial pellet was lysed by protease to release target DNA. Then, DNA-MSB complexes were formed as the tube was intermittently rotated and distributed uniformly onto the iron wire netting inside the Halbach cylinder magnet. Finally, the purified DNA was amplified using RAA and quantitatively detected by the CRISPR-Cas12a assay. Results: This biosensor could quantitatively detect Salmonella in spiked milk samples in 75 min, with a lower detection limit of 6 CFU/mL. The fluorescent signal of 102 CFU/mL Salmonella Typhimurium was over 2000 RFU, while 104 CFU/mL Listeria monocytogenes, Bacillus cereus, and E. coli O157:H7 were selected as non-target bacteria and had signals less than 500 RFU (same as the negative control). Conclusions: This lab-on-a-tube biosensor integrates cell lysis, DNA extraction, and RAA amplification in one 15 mL tube to simplify the operation and avoid contamination, making it suitable for low-concentration Salmonella detection.
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