Loop-mediated isothermal amplification (LAMP) is a simple, highly sensitive, rapid, and low-cost nucleic acid amplification method that holds great potential for point-of-care (POC) diagnostics. Differing from the conventional polymerase chain reaction technique, LAMP is performed at a single temperature, which eliminates the use of expensive thermal cyclers. However, a major limitation of LAMP is its detection methods which typically rely on turbidity and non-specific dyes for colorimetric determination. The results of these methods are subjective when observed with the naked eye and can lead to false-positive results. To circumvent many of these shortcomings, a method coupling molecular beacons (MBs) with digital droplet LAMP (MB-ddLAMP) has been developed as an alternative method to perform absolute quantification of wild-type and mutant DNA samples. MBs are single-stranded hairpin-shaped oligonucleotide probes that emit fluorescence only when forming a duplex with a complementary single-stranded DNA target. MB-ddLAMP consists of thousands of microdroplets that are emulsified from a bulk ddLAMP mixture. Based on the random distribution of DNA that partitions into the droplets, each droplet can be considered as a single reaction chamber and the droplets can be classified into positive and negative results based on the fluorescence intensity of the droplets. Commercial droplet generators required to perform ddLAMP cost over 100,000 USD, thereby severely limiting the use of ddLAMP in many laboratories as well as in POC diagnostics. In this presentation, we demonstrate a simple and low-cost setup using a stereolithography 3D-printed droplet generator system which costs less than 900 USD for the entire system. In addition, the droplet generation process can produce nearly 10,000 homogenous droplets at diameters of approximately 120 μm from 20 μL LAMP mixture. The droplet size can be easily customized to range from diameters of 120-500 μm. Using this system, we demonstrate single nucleotide polymorphism detection of the OmpW DNA sequence with MB-ddLAMP.

Molecular beacons coupled with digital droplet loop-mediated isothermal amplification using 3D-printed droplet generator for single nucleotide polymorphism detection

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ACS Fall 2022

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