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

作　　者：Jinrong Wang Guowei Song Yue Ming Jing Pan Ruiqing Zhang Guohao Fan Xinxin Shen Xuejun Ma Lixin Li 

机构地区：[1]Shijiazhuang People’s Hospital,Shijiazhuang,Hebei,050051,China [2]Qiqihar Medical University,Qiqihar,Heilongjiang,161000,China [3]NHC Key Laboratory of Medical Virology and Viral Diseases,National Institute for Viral Disease Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing,102206,China

出　　处：《Infectious Medicine》2022年第2期73-80,共8页感染医学（英文）

基　　金：supported by grants from the Youth Foundation of Academician Hou Yunde[grant number 2019HYDQNJJ03];China Mega-Projects for Infec-tious Disease[grant number 2017ZX10302301-004-002].

摘　　要：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.

关 键 词：Nucleic acid amplification enhancer BETAINE PULLULAN RAA Long-fragment 

分 类 号：Q789[生物学—分子生物学]