| Info. | Vol.16 - No.4 (2022.12.20) |
|---|---|
| Title | A Lateral Flow Assay for Nucleic Acid Detection Based on Rolling Circle Amplification Using Capture LigandModified Oligonucleotides |
| Authors | Ha Neul Lee1,2, Juhee Lee1, Yoo Kyung Kang1, Joo Hoon Lee2, Seungju Yang1, Hyun Jung Chung1,2
*Hyun Jung Chung hyunjc@kaist.ac.kr |
| Institutions | 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea
2Graduate School of Nanoscience and Technology, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea |
| Abstract | We introduce a lateral flow assay (LFA) integrated with a modified isothermal nucleic acid amplification procedure for rapid and simple genetic testing. Padlock probes specific for the target DNA were designed for ligation, followed by rolling circle amplification (RCA) using capture ligand-modified oligonucleotides as primers. After hybridization with detection linker probes, the amplified target DNA is flowed through an LFA membrane strip for binding of gold nanoparticles as the substrate for colorimetric detection. We established and validated the “RCA-LFA” method for detection of mecA, the antibiotic resistance gene for methicillin-resistant Staphylococcus aureus (MRSA). The assay was optimized using various concentrations of primers and probes for RCA and LFA, respectively. The sensitivity was determined by performing RCA-LFA using various amounts of mecA target DNA, showing a detection limit of ~ 1.3 fmol. The specificity of the assay was examined using target DNAs for other resistance genes as the controls, which demonstrated positive detection signals only for mecA DNA, when added either individually or in combinations with the control targets. Furthermore, applying the RCA-LFA method using specifically designed probes for RNA-dependent RNA polymerase (RdRp) and receptor binding domain (RBD) gene for SARS-CoV-2, which demonstrated feasibility of the method for viral gene targets. The current method suggests a useful platform which can be universally applied for various nucleic acid targets, allowing rapid and sensitive diagnosis at point-of-care. |
| Keyword | Lateral flow assay, Rolling circle amplification, Rapid diagnosis, Genetic testing, Point-of-care |
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