Abstract
In this work, we report digital loop-mediated isothermal amplification (LAMP) or reverse-transcription LAMP (RT-LAMP) on a commercial membrane, without the need for complex chip fabrication or use of specialized equipment. Due to the pore size distribution, the theoretical error for digital LAMP on these membranes was analyzed, using a combination of Random Distribution Model and Multivolume Theory. A facile peel-off process was developed for effective droplet formation on the commercial track-etched polycarbonate (PCTE) membrane. Each pore functions as an individual nanoreactor for single DNA amplification. Absolute quantification of bacteria genomic DNA was realized with a dynamic range from 11 to 1.1 × 105 copies/μL. One-step digital RT-LAMP was also successfully performed on the membrane for the quantification of MS2 virus in wastewater. With the introduction of new probes, the positive pores can be easily distinguished from negative ones with 100 times difference in fluorescence intensities. Finally, the cost of a disposable membrane is less than $0.10/piece, which, to the best of our knowledge, is the most inexpensive way to perform digital LAMP. The membrane system offers opportunities for point-of-care users or common laboratories to perform digital quantification, single cell analysis, or other bioassays in an inexpensive, flexible, and simplified way.
Highlights
Digital PCR has become a promising technologies for absolute quantification of nucleic acid without need of calibration curves.[1]
The track-etched membrane is a type of commercial membranes which contains a high density of micro/nanopores with uniform pore sizes, ranging from 10 nm to 30 μm.[34]
104 uniform picoliter droplets could be generated on a single membrane within 1 min
Summary
Digital PCR (dPCR) has become a promising technologies for absolute quantification of nucleic acid without need of calibration curves.[1]. The overlapped pores have almost double volume (see Figure S1), resulting in an error for the digital nucleic acid quantification. Considering the dynamic range (10−105 copy/μL) of our membrane system, using an average pore volume for the calculation of DNA concentrations is more reliable.
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