Establishment of Dual Hairpin Ligation-Induced Isothermal Amplification for Universal, Accurate, and Flexible Nucleic Acid Detection

Isothermal amplifications have found their potentials in applications of portable nucleic acid diagnostics. However, there are still several certain deficiencies existing in the current amplification methods, including high false-positive signals, limited range of targets, difficult primer design, and so forth. Here, we report an effective solution via the development of dual hairpin ligation-induced isothermal amplification (DHLA) consisting of (1) the formation of a dual hairpin probe (DHP) based on sequence specific hybridization and ligation and (2) exponential isothermal amplification of DHP in the presence of polymerase and primers. Taking both microRNA and virus RNA as model targets, DHLA is proven to be accurate, flexible, and applicable to most deoxyribonucleic acid and ribonucleic acid targets ranging from similar to 20 to hundreds of nt. The detection limit is down to the similar to aM level without a false-positive signal. More importantly, the whole detection can be directly applied to a new target via a slight change in the DHP sequence, without redesigning the primer set. This unique property not only simplifies the process for new reaction development but also enables flexible multiprobe strategies to achieve antidegradation analysis.

Automated summary

Dual hairpin ligation-induced isothermal amplification (DHLA) is an effective nucleic acid amplification method, capable of accurately and flexibly detecting various targets with a detection limit down to the aM level without false-positive signals. This method allows direct detection of new targets by simply changing the probe sequence, without the need to redesign the primer set.