Recombinase polymerase amplification (RPA) is an isothermal amplification technique. Because of its short detection cycle and high specificity, it has been applied in various fields. However, the design of probe on the efficiency of RPA is not well understood and the effect of sequence mismatches of oligonucleotides on the performance of RPA is rarely discussed. In this study, we found that different primers with the same probe have a slight effect on the efficiency of fluorescent RPA, and different probes with the same amplified region have a great influence on the efficiency of fluorescent RPA. We summarized the design rules of probes suitable for fluorescent RPA by analyzing the experimental data. The rule is that the best distance between fluorescent groups in the probe is 1-2 bases, and the G content should be reduced as far as possible. In addition, we verified this rule by designing a series of probes. Furthermore, we found the base mismatches of the probe had a significant effect on RPA, which can lead to false positives and can change the amplification efficiency. However, 1-3 mismatches covering the center of the primer sequence only affect the amplification efficiency of RPA, not its specificity. And with an increase in the number of primer mismatches, the efficiency of RPA will decrease accordingly. This study suggests that the efficiency of fluorescent RPA is closely related to the probe. We recommend that when designing a fluorescent probe, one must consider the presence of closely related non-targets and specific bases.
Keywords: Design rule; Efficiency; Mismatch; Probe; RPA.