Primer design strategy for denaturation bubble-mediated strand exchange amplification

Denaturation bubble-mediated strand exchange amplification (SEA) is a novel, rapid isothermal nucleic acid amplification has been applied for point-of-care molecular diagnostic in food safety, meat adulteration, forest disease and animal disease. Nevertheless, the absence of specialized strategy for SEA primers design led to long-time of primer screening progress before SEA reaction execution, which would largely increase the time consuming when SEA is utilized for detecting other new targets. In this present work, we investigated the impact of the following primers' attributes on SEA efficiency, including Tm value, 3' end G/C content, self-complementary and 3' complementary, according to which we demonstrated that optimal Tm value and reaction temperature were all 61 °C, while 3'-terminal nucleotide should be G/C, as the SEA reaction induced by the primers possessing these attributes exhibited significantly lower threshold time (Tt) value. Moreover, self-complementary and 3' complementary of primers should be avoided. Besides, we also discussed the consideration priority order of these factors, which was self-complementary and 3' complementary, Tm value and 3' end G/C content in turn. Because the SEA primer design strategy is first presented, our work will greatly promote the application of SEA in point-of-care test.