A paper based microfluidic platform combining LAMP-CRISPR/Cas12a for fluorometric detection of nucleic acids

Abstract

Nucleic acid isothermal amplification methods are advantageous for point-of-care (POC) diagnostics because of their precision, sensitivity, and low power requirements. Loop-mediated isothermal amplification (LAMP) is an established method, renowned for its nucleic acid amplification efficiency and robust amplification of semi purified target nucleic acids. However, LAMP may be prone to non-specific amplification causing false positives and therefore fails to replace PCR as the gold standard method in clinical testing. We show that LAMP combined with clustered regularly interspaced short palindromic repeats (CRISPR) technology is an effective alternative for overcoming the limitations of LAMP alone. Nucleic acids are first isothermally pre-amplified to enrich for targets, then specific amplification detection signals are generated by sequences of RNA guided recognition of amplicons. We are the first to demonstrate a paper based microfluidic system for detecting pathogen nucleic acids in samples by combining the power of LAMP and CRISPR technology. We show that although LAMP may produce non-specific amplification, the possibility of detecting a false positive can be eliminated by combining LAMP with CRISPR based detection. We demonstrate that a paper based microfluidic platform has the potential to compete with the conventional Nucleic acid testing (NAT) technologies not only in terms of robustness but also in terms of cost and complexity.