Amplification Bias-Free Sequence-Generic Exponential Amplification Reaction

Despite the unique advantage of the isothermal exponential amplification reaction (EXPAR) for the rapid detection of short nucleic acids, it severely suffers from the drawback of sequence-dependent amplification bias, mainly arising from the secondary structures of the EXPAR template under the commonly used reaction temperature (55 °C). As such, the limits of detection (LOD) for different target sequences may vary considerably from aM to nM. Here we report a sequence-generic exponential amplification reaction (SG-EXPAR) that eliminates sequence-dependent amplification bias and achieves similar amplification performance for different targets with generally sub-fM LODs. The assay innovatively employs a thermophilic nicking enzyme that allows SG-EXPAR to work efficiently at higher temperatures (60–70 °C) while eliminating the secondary structures of the templates, which is the basis for eliminating the amplification bias. Furthermore, we increased the probability of trigger/template binding through rational modification of the locked nucleic acids and template optimization, further ensuring the high amplification efficiency for various targets. According to these critical principles, we have developed an automated design platform that allows nonspecialists to obtain the optimal SG-EXPAR template for any desired sequence. The robust performance of the proposed methodology was demonstrated by quantifying microRNA, SARS-CoV-2, monkeypox virus, and HPV B19 at the 1 fM level without sequence screening. SG-EXPAR significantly expands the potential applications of EXPAR and facilitates the development of reliable point-of-care nucleic acid assays.