Digital isothermal quantification of nucleic acids via simultaneous chemical initiation of recombinase polymerase amplification reactions on SlipChip

Abstract

In this paper, digital quantitative detection of nucleic acids was achieved at the single-molecule level by chemical initiation of over one thousand sequence-specific, nanoliter isothermal amplification reactions in parallel. Digital polymerase chain reaction (digital PCR), a method used for quantification of nucleic acids, counts the presence or absence of amplification of individual molecules. However, it still requires temperature cycling, which is undesirable under resource-limited conditions. This makes isothermal methods for nucleic acid amplification, such as recombinase polymerase amplification (RPA), more attractive. A microfluidic digital RPA SlipChip is described here for simultaneous initiation of over one thousand nL-scale RPA reactions by adding a chemical initiator to each reaction compartment with a simple slipping step after instrument-free pipet loading. Two designs of the SlipChip, two-step slipping and one-step slipping, were validated using digital RPA. By using the digital RPA SlipChip, false-positive results from preinitiation of the RPA amplification reaction before incubation were eliminated. End point fluorescence readout was used for "yes or no" digital quantification. The performance of digital RPA in a SlipChip was validated by amplifying and counting single molecules of the target nucleic acid, methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA. The digital RPA on SlipChip was also tolerant to fluctuations of the incubation temperature (37-42 °C), and its performance was comparable to digital PCR on the same SlipChip design. The digital RPA SlipChip provides a simple method to quantify nucleic acids without requiring thermal cycling or kinetic measurements, with potential applications in diagnostics and environmental monitoring under resource-limited settings. The ability to initiate thousands of chemical reactions in parallel on the nanoliter scale using solvent-resistant glass devices is likely to be useful for a broader range of applications.

Figure 1

RPA amplification of MRSA genomic DNA (5 pg/μL) in a well plate at 25°C. Triplicate curves (green lines) show that gDNA template was amplified at room temperature. The control experiment without template (orange line) and the control experiment without magnesium acetate (Mg(OAc)₂, blue line) show no amplification.

Figure 2

Schematic drawing of the two-step SlipChip for digital RPA. A) Top plate of the SlipChip. A zoomed in schematic drawing shows the geometry of Type I, Type II and satellite wells. B) Bottom plate of the SlipChip. C) Assembly of top and bottom plates to establish the first continuous fluidic path of Type I wells. D) Loading of the first reagent, Reaction Mixture 1 (red). E) Slipping breaks the first fluidic path and compartmentalizes the loaded reagent. At the same time, the second fluidic path is formed by connecting Type II wells. The second reagent, Reaction Mixture 2 (light blue), is loaded through a second inlet. F) A second slipping step compartmentalizes Reaction Mixture 2 into the Type II wells and overlaps the Type II wells with the Type I wells. The two reagents are mixed within the reaction compartments. G) Photograph shows the entire digital RPA SlipChip next to a US quarter for size comparison. H, I, J) Food dyes were loaded into the SlipChip to demonstrate loading and mixing. H) Zoomed in view of Type II wells for Control 2 (no template), loaded with blue food dye. I) Zoomed in view of reaction wells (overlapping Type I and Type II wells) containing mixed blue and orange food dye (green). H) Zoomed in view of Type I wells for Control 1 (no magnesium acetate), loaded with orange food dye. K,L,M,N) Experiments with food dye demonstrate the procedures described in D, E, F.

Figure 3

Fluorescence microphotographs and linescans of RPA on the SlipChip before and after incubation at 39 °C. A–B) Negative (left) and positive (right) sample wells: (A) before incubation, the fluorescence intensity in both wells is the same. (B) After incubation, the integrated fluorescence intensity in the positive well (right) is significantly higher compared to the negative well (left). C–D) Control well 1, containing no magnesium acetate, before (C) and after (D) incubation shows no significant increase in fluorescence intensity. E–F) Control well 2, containing no gDNA template, before (E) and after (F) incubation also shows no significant increase in fluorescence intensity.

Figure 4

Digital RPA on the SlipChip with different concentration of MRSA gDNA. A–E) Digital RPA on the SlipChip with a serial dilution of target DNA template ranging from 1:10 to 1:10⁵ of a 5 ng/μL stock solution. F) Control, no wells showed positive signal when no target DNA was loaded.

Figure 5

Quantified results of digital RPA on the SlipChip. Experimental average of the number of positive wells was plotted as a function of the dilution of the MRSA gDNA sample. Error bars represent standard deviation of the experiment (n=3). The black solid line represents the Poisson distribution of the calculated stock concentration from fitting the data from the 1:10⁵, 1:10⁴, and 1:10³ dilutions of template. Gray dash lines represent the 95% confidence interval for the fitted Poisson distribution.

Figure 6

SlipChip for one-step digital RPA. A–C) Schematic drawings of the SlipChip: A) Assembly of top (solid) and bottom (dashed) plates to establish the continuous fluidic path for both Type I wells and Type II wells. B) The first solution, Reaction Mixture 1 (red), and second solution, Reaction Mixture 2 (blue), are introduced simultaneously into the SlipChip. C) Slipping breaks both fluidic paths and compartmentalizes the loaded reagent. At the same time, the Type I wells are overlaid with Type II wells to initiate the reaction. D, E) Microphotographs showing food dyes loaded into the SlipChip to demonstrate loading and mixing. F) Zoomed-in fluorescent image of a fraction of digital RPA on one-step SlipChip with a 1:10⁴ dilution of MRSA gDNA template after incubation at 39°C.

Figure 7

A) Comparing on-chip mixing (no pre-initiation) to pre-initiation with magnesium acetate on the two-step digital RPA SlipChip. The sample with pre-initiation with magnesium acetate prior to compartmentalization shows a higher fraction of positive wells, indicating that compartmentalization prior to the addition of magnesium acetate is crucial to achieve accurate digital RPA. B) Comparing two-step digital RPA, one-step digital RPA and digital PCR. Samples containing MRSA gDNA at the same dilution (1:10⁴) were quantified using two-step digital RPA (as in Figure 4) (left, n=3), one-step digital RPA (as in Figure 6) (middle, n=5), and digital PCR (right, n=3) on the RPA SlipChip. Error bars represent standard deviation.

Figure 8

RPA two-step SlipChip for amplification of MRSA gDNA with incubation at different temperatures. A–C) Representative fluorescent images of RPA for MRSA gDNA with dilution of 1:10⁴ at 37°C (A), 39°C (B), and 42°C (C). D) Histogram showing number of positive wells from RPA on the SlipChip at different incubation temperatures. Error bars represent standard deviation of the experiment (p > 0.2, n ≥ 4).