|REN, WEN - Purdue University|
|AHMAD, SAEED - Purdue University|
|IRUDAYARAJ, JOSEPH - Purdue University|
Submitted to: ACS Omega
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/31/2021
Publication Date: 4/14/2021
Citation: Ren, W., Ahmad, S., Irudayaraj, J. 2021. 16S rRNA monitoring point-of-care magnetic focus lateral flow sensor. 6:11095-11102. https://doi.org/10.1021/acsomega.1c01307.
Interpretive Summary: The detection and identification of pathogenic bacteria is critical for human health, environmental, and food safety monitoring. This study describes a highly sensitive strategy for naked eye detection of pathogenic bacteria relevant to infections, human health, and food safety monitoring via the detection of specific DNA sequences. The method uses a magnetic focus lateral flow sensor (mLFS) platform (similar technology to a home pregnancy test using a magnet and other components to improve sensitivity). The method developed was demonstrated in model system using a specific gene sequence from pathogen Escherichia coli O157:H7 (within the 16S rRNA gene). The use of magnetic focusing improved the sensitivity of the method nearly 100,000 compared to other similar lateral flow sensors. Our approach highlights the possibility of extending this concept to screen specific DNA sequences for the monitoring of infectious pathogens implicated in a range of foodborne diseases.
Technical Abstract: The detection and profiling of pathogenic bacteria is critical for human health, environmental, and food safety monitoring. Herein, we propose a highly sensitive colorimetric strategy for naked eye screening of 16S ribosomal RNA (16S rRNA) from pathogenic agents relevant to infections, human health, and food safety monitoring with a magnetic focus lateral flow sensor (mLFS) platform. The method developed was demonstrated in model 16S rRNA sequences of the pathogen Escherichia coli O157:H7 to detect as low as 1 fM of targets, exhibiting a sensitivity improved by ~5 × 105 times compared to the conventional GNP-based colorimetric lateral flow assay used for oligonucleotide testing. Based on the grayscale values, semi-quantitation of up to 1 pM of target sequences was possible in ~45 min. The methodology could detect the target 16S rRNA from as low as 32 pg/mL of total RNA extracted from pathogens. Specificity was demonstrated with total RNA extracted from E. coli K-12 MG1655, Bacillus subtilis (B. subtilis), and Pseudomonas aeruginosa (P. aeruginosa). No signal was observed from as high as 320 pg/mL of total RNA from the nontarget bacteria. The recognition of target 16S rRNA from 32 pg/mL of total RNA in complex matrices was also demonstrated. The proposed mLFS method was then extended to monitoring B. subtilis and P. aeruginosa. Our approach highlights the possibility of extending this concept to screen specific nucleic acid sequences for the monitoring of infectious pathogens or microbiome implicated in a range of diseases including cancer.