Enhancement of a recombinase-aided amplification assay using betaine and pullulan

Abstract

Background: Nucleic acid amplification enhancers suitable for use in a recombinase-aided amplification (RAA) assay were studied for the first time, and amplification of a long-fragment (509 bp) was initially explored.

Methods: Using recombinant plasmids and clinical samples, RAA fluorescence and basic methods were used to evaluate the efficacy. The fluorescence method was evaluated by threshold time and fluorescence value, and the basic method was characterized by 2% agarose gel electrophoresis.

Results: Taking a previously established RAA assay for HPV18 as an example, we demonstrated that the addition of 0.2 M, 0.4 M, and 0.6 M betaine and 10% pullulan could enhance the RAA. The new RAA assays with betaine and pullulan were named B-RAA and P-RAA, respectively. Using the B-RAA and P-RAA fluorescence methods, the threshold time values could be shortened by 1.72-2.32 minutes and 2.60 minutes, respectively, and the fluorescence values could be enhanced by 8847.25-9094.37 mv and 5250 mv, respectively. Using the basic method, the sensitivity could be increased 10-fold. We successfully amplified a long-fragment of 509 bp using a P-RAA assay with a sensitivity of 10² copies/µL (compared with 10³ copies/µL in the RAA assay).

Conclusions: Thus, we concluded that betaine and pullulan are effective additives to enhance the sensitivity of RAA assays.

Keywords: Betaine; Long-fragment; Nucleic acid amplification enhancer; Pullulan; RAA.

Graphical abstract

Fig. 1

(A) B-RAA fluorescence amplification results of 10 copies/µL of HPV18 plasmid with different concentrations of betaine. (B) B-RAA basic amplification results of 10 copies/µL of HPV18 plasmid with different concentrations of betaine. Lane M: Marker; Lane 1–7: negative control, betaine-free, 0.2 M betaine, 0.4 M betaine, 0.6 M betaine, 0.8 M betaine, and 1.0 M betaine.

Fig. 2

(A) P-RAA fluorescence amplification results of 10 copies/µL of HPV18 plasmid with different concentrations of pullulan. (B) P-RAA basic amplification results of 10 copies/µL of HPV18 plasmid with different concentrations of pullulan. Lane M: Marker; Lane 1–7: negative control, pullulan-free, 2% pullulan, 4% pullulan, 6% pullulan, 8% pullulan, and 10% pullulan.

Fig. 3

(A) RAA fluorescence long-fragment amplification results of 10²–10⁴ copies/µL of the HPV18 plasmid using the original RAA, B-RAA, and P-RAA assays. (B) RAA basic long-fragment amplification results of 10²–10⁴ copies/µL of HPV18 plasmid with the original RAA, B-RAA, and P-RAA assays. Lane 1: negative control. Lanes 2–4: Amplification of 10²–10⁴ copies/µL of HPV18 plasmid by the original RAA. Lanes 5–7: Amplification of 10²–10⁴ copies/µL of HPV18 plasmid by B-RAA. Lanes 8–10: Amplification of 10²–10⁴ copies/µL of HPV18 plasmid by P-RAA.

Fig. 4

The fluorescence values of a 509 bp long fragment amplification of 10²–10⁴ copies/µL of the HPV18 plasmid using the original RAA, B-RAA, and P-RAA assays. The data are shown for eight replicates.

Fig. 5

The TT values of a 509 bp long fragment amplification of 10²–10⁴ copies/µL of the HPV18 plasmid using the original RAA, B-RAA, and P-RAA assays. (Note: No bar corresponds to negative, not 0). The data are shown for eight replicates.