Measuring Fate and Rate of Single‐Molecule Competition of Amplification and Restriction Digestion, and Its Use for Rapid Genotyping Tested with Hepatitis C Viral RNA

Abstract

We experimentally monitored, at the single-molecule level, the competition among reverse transcription, exponential amplification (RT-LAMP), and linear degradation (restriction enzymes) starting with Hepatitis C viral RNA molecules. We found significant heterogeneity in the rate of single-molecule amplification; introduction of the restriction enzymes affected both the rate and the “fate” (the binary outcome) of single-molecule amplification. While end-point digital measurements were primarily sensitive to changes in fate, the bulk real-time kinetic measurements were dominated by the rate of amplification of the earliest molecules, and not sensitive to fate of the rest of the molecules. We showed how this competition of reactions can be used for rapid HCV genotyping with either digital or bulk readout. This work advances our understanding of single-molecule dynamics in reaction networks and may help bring genotyping capabilities out of clinical labs and into limited-resource settings.

Digital Quantification of DNA Replication and Chromosome Segregation Enables Determination of Antimicrobial Susceptibility after only 15 Minutes of Antibiotic Exposure

“…We anticipate that digital isothermal amplification chemistries will replace dPCR in dAST. [15a,21] When integrated with sample preparation [22] and combined with simple readouts, [23] we envision that digital quantification will establish a new paradigm in rapid point of care AST.…”

Droplet and Microchamber‐Based Digital Loop‐Mediated Isothermal Amplification (dLAMP)

“…After compartmentalizing and heating, positive LAMP reactions are performed in the compartments when the target nucleic acids are present. To visualize these positive LAMP reactions and calculate the portion of these positive reactions, a fluorescence dye, such as calcein, is typically added into the LAMP mixtures to indicate the amplification results. In a typical positive run, the LAMP reaction byproduct, pyrophosphate, binds with calcein‐bond manganese ions, producing free calcein molecules which bind with magnesium ion and emit strong fluorescence ( Figure a1); In a negative run, calcein binds with manganese ions and emits dim fluorescence (Figure a2) .…”

“…The size, uniformity and number of the microchambers are all crucial for dLAMP, since they determine the total number of the compartments of the LAMP mixtures, and affect the dLAMP's sensitivity and dynamic range, which is defined as the detectable concentration ranges of a target nucleic acid, depending on the number of partitions analyzed, and the resolution, which is defined as the ability of differentiating positive results from negative results in a linear separation . We summarize the chamber size and number, together with the consequent sensitivity and dynamic range of the dLAMP in Table 1 .…”

“…In the loading step of LAMP mixtures, the two plates are assembled and aligned to introduce the LAMP mixtures, as shown in Figure c1. Afterward, the plates of the chip are moved relative to one another, confining the LAMP mixtures into individual microchambers without cross‐contamination, as shown in Figure c2 . This approach avoids the complicated hydrodynamic analysis and the fabrication process of multilayer device.…”

The pumping lid: investigating multi-material 3D printing for equipment-free, programmable generation of positive and negative pressures for microfluidic applications

“…This is a good test because loading SlipChip devices requires control of the inlet pressure within a defined range, 25 and SlipChips are intended to be used in limited resource settings (LRS) by untrained users. [26][27][28][29] First, we tested the pumping lid on a SlipChip designed for a digital nucleic acid detection assay 26 (Fig. 6A), pumping a total of 5 μL of solution with 0.03 atm pressure (eqn (1)).…”