Since its introduction in 1985, polymerase chain reaction (PCR) has greatly promoted the development of molecular biology research and disease diagnosis and also significantly promoted the identification of novel genes and other biomedical advances. However, for the detection of single-base mutations, there is still an urgent need for nucleic acid amplification and detection methods that accurately identify single-base differences. In 1988, Landegren demonstrated that a ligase-based reaction could distinguish the single-base mutated β-globin gene (βS) in patients with sickle-cell anemia from the normal β-globin gene (βA) [1]. Similar to PCR, which requires amplification of DNA molecules through multiple temperature cycles, ligase-catalyzed ligation reaction can also be cycled through high-temperature denaturation, annealing, and ligation steps to achieve signal or target amplification. In 1989, Wu further designed an amplification method for multiple rounds of ligation reaction, which has the ability of exponential amplification of DNA and realizes single-base differentiation of β-globin gene [2]. However, the T4 DNA ligase used at the time was not heat-resistant, resulting in the enzyme losing activity at the thermal denaturation stage of each cycle, and the ligase had to be frequently replenished to maintain the reaction. In order to overcome this problem, in 1991, Barany applied heat-resistant DNA ligase in ligase chain reaction (LCR) for the detection of single-base mutation of β-globin gene [3], avoiding the tedious steps of constantly replenishing enzymes during the thermal cycle and promoting the wide application of LCR.