Precise definition of starting time by capillary-based chemical initiation of digital isothermal DNA amplification

Abstract

Digital polymerase chain reaction is a sensitive and reproducible method to assess the presence or absence of the amplification of individual target molecules confined in isolated compartments; this is used for the quantification of nucleic acids. In this paper, the precise definition of the start time of recombinase polymerase amplification (RPA) is proposed to achieve the digital quantification of nucleic acids at the single molecule level. RPA is a sequence-specific isothermal amplification method. Since the reaction will start immediately albeit slowly at room temperature following the addition of the chemical initiator (magnesium acetate), the number of false positives in digital RPA is increased if all reagents are mixed prior to compartmentalization. A capillary-based setup is described here to control the initiation of RPA reactions by encapsulating the chemical initiator to each reaction compartment using shear force when passed through a cross connector. Thousands of independent compartments are generated. The performance of digital droplet RPA (ddRPA) was validated by counting the positive application results of target molecules (Avian virus DNA) confined in the partitions. The ddPRA capillary-based setup provides a simple nucleic acid quantification method without thermal cycling. Potential applications in clinical and academic research under resource-limited settings can be envisaged. The ability to initiate chemical reaction compartments by the encapsulation of a chemical initiator using similar capillaries can be applied to a broader range of applications.