Enhancement of a recombinase-aided amplification assay using betaine and pullulan
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 bas...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-06-01
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| Series: | Infectious Medicine |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2772431X22000211 |
| _version_ | 1797345845997207552 |
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| author | Jinrong Wang Guowei Song Yue Ming Jing Pan Ruiqing Zhang Guohao Fan Xinxin Shen Xuejun Ma Lixin Li |
| author_facet | Jinrong Wang Guowei Song Yue Ming Jing Pan Ruiqing Zhang Guohao Fan Xinxin Shen Xuejun Ma Lixin Li |
| author_sort | Jinrong Wang |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-08T11:24:37Z |
| format | Article |
| id | doaj.art-4e211743fc8543b5a030532e01d8b8ce |
| institution | Directory Open Access Journal |
| issn | 2772-431X |
| language | English |
| last_indexed | 2024-03-08T11:24:37Z |
| publishDate | 2022-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Infectious Medicine |
| spelling | doaj.art-4e211743fc8543b5a030532e01d8b8ce2024-01-26T05:35:58ZengElsevierInfectious Medicine2772-431X2022-06-01127380Enhancement of a recombinase-aided amplification assay using betaine and pullulanJinrong Wang0Guowei Song1Yue Ming2Jing Pan3Ruiqing Zhang4Guohao Fan5Xinxin Shen6Xuejun Ma7Lixin Li8Shijiazhuang People's Hospital, Shijiazhuang, Hebei, 050051, ChinaShijiazhuang People's Hospital, Shijiazhuang, Hebei, 050051, ChinaQiqihar Medical University, Qiqihar, Heilongjiang, 161000, ChinaShijiazhuang People's Hospital, Shijiazhuang, Hebei, 050051, ChinaNHC 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, ChinaNHC 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, ChinaNHC 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; Corresponding authors.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; Corresponding authors.Shijiazhuang People's Hospital, Shijiazhuang, Hebei, 050051, China; Corresponding authors.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.http://www.sciencedirect.com/science/article/pii/S2772431X22000211Nucleic acid amplification enhancerBetainePullulanRAALong-fragment |
| spellingShingle | Jinrong Wang Guowei Song Yue Ming Jing Pan Ruiqing Zhang Guohao Fan Xinxin Shen Xuejun Ma Lixin Li Enhancement of a recombinase-aided amplification assay using betaine and pullulan Infectious Medicine Nucleic acid amplification enhancer Betaine Pullulan RAA Long-fragment |
| title | Enhancement of a recombinase-aided amplification assay using betaine and pullulan |
| title_full | Enhancement of a recombinase-aided amplification assay using betaine and pullulan |
| title_fullStr | Enhancement of a recombinase-aided amplification assay using betaine and pullulan |
| title_full_unstemmed | Enhancement of a recombinase-aided amplification assay using betaine and pullulan |
| title_short | Enhancement of a recombinase-aided amplification assay using betaine and pullulan |
| title_sort | enhancement of a recombinase aided amplification assay using betaine and pullulan |
| topic | Nucleic acid amplification enhancer Betaine Pullulan RAA Long-fragment |
| url | http://www.sciencedirect.com/science/article/pii/S2772431X22000211 |
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