@article{Wang2022, 
author = {Jinrong Wang and Guowei Song and Yue Ming and Jing Pan and Ruiqing Zhang and Guohao Fan and Xinxin Shen and Xuejun Ma and Lixin Li},
title = {Enhancement of a recombinase-aided amplification assay using betaine and pullulan},
year = {2022},
journal = {Infectious Medicine},
volume = {1},
number = {2},
pages = {73-80},
keywords = {Nucleic acid amplification enhancer, Betaine, Pullulan, RAA, Long-fragment},
url = {https://www.sciopen.com/article/10.1016/j.imj.2022.06.002},
doi = {10.1016/j.imj.2022.06.002},
abstract = {BackgroundNucleic 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.MethodsUsing 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.ResultsTaking 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).ConclusionsThus, we concluded that betaine and pullulan are effective additives to enhance the sensitivity of RAA assays.}
}