In 2003, David J. Galas et al. first reported the Isothermal Exponential Amplification Reaction (EXPAR) [1]. EXPAR utilizes DNA templates, deoxyribonucleotide triphosphate (dNTPs), DNA polymerase, and nicking endonuclease to achieve exponential amplification of target sequences. The basic principle of a classical single-template EXPAR is shown in Fig. 5.1 [2]. First, an amplification template (X′-X′) is designed based on the target sequence (Target). The 3′-end and 5′-end of the amplification template are sequences X′ (blue) with the same bases, which are complementary to the target. The intermediate sequence (black) of the amplification template is the nicking recognition sequence of the nicking endonuclease (NEase). When the target is present, it hybridizes with the 3′-end of the amplification template (i). By the catalytic action of DNA polymerase, the target sequence undergoes an elongation reaction along the template, resulting in the formation of double-stranded DNA (dsDNA) (ii). The NEase specifically recognizes the nicking recognition sequence on dsDNA (Nicking sequence) and creates a cut on the upper strand (iii). The DNA polymerase continues the elongation reaction at the incision site, which releases short DNA (iv) through strand displacement of the polymerase and forms new dsDNA. The new dsDNA undergoes continuous enzymatic cleavage, elongation, and strand displacement under the joint action of the NEase and polymerase, continuously releasing new short DNA and forming a linear amplification mechanism.