A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane
Nucleic acid quantification, allowing us to accurately know the copy number of target nucleic acids, is significant for diagnosis, food safety, agricultural production, and environmental protection. However, current digital quantification methods require expensive instruments or complicated microflu...
| Main Authors: | , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-03-01
|
| Series: | Biosensors |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2079-6374/14/3/139 |
| _version_ | 1797241846047440896 |
|---|---|
| author | Yanju Chen Yuanyuan Zhu Cheng Peng Xiaofu Wang Jian Wu Huan Chen Junfeng Xu |
| author_facet | Yanju Chen Yuanyuan Zhu Cheng Peng Xiaofu Wang Jian Wu Huan Chen Junfeng Xu |
| author_sort | Yanju Chen |
| collection | DOAJ |
| description | Nucleic acid quantification, allowing us to accurately know the copy number of target nucleic acids, is significant for diagnosis, food safety, agricultural production, and environmental protection. However, current digital quantification methods require expensive instruments or complicated microfluidic chips, making it difficult to popularize in the point-of-care detection. Paper is an inexpensive and readily available material. In this study, we propose a simple and cost-effective paper membrane-based digital loop-mediated isothermal amplification (LAMP) method for nucleic acid quantification. In the presence of DNA fluorescence dyes, the high background signals will cover up the amplicons-formed bright spots. To reduce the background fluorescence signals, a quencher-fluorophore duplex was introduced in LAMP primers to replace non-specific fluorescence dyes. After that, the amplicons-formed spots on the paper membrane can be observed; thus, the target DNA can be quantified by counting the spots. Take <i>Vibrio parahaemolyticus</i> DNA detection as an instance, a good linear relationship is obtained between the light spots and the copy numbers of DNA. The paper membrane-based digital LAMP detection can detect 100 copies target DNA per reaction within 30 min. Overall, the proposed nucleic acid quantification method has the advantages of a simple workflow, short sample-in and answer-out time, low cost, and high signal-to-noise, which is promising for application in resourced limited areas. |
| first_indexed | 2024-04-24T18:29:48Z |
| format | Article |
| id | doaj.art-1f8683d590904d59bf814643f54bee2c |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-04-24T18:29:48Z |
| publishDate | 2024-03-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-1f8683d590904d59bf814643f54bee2c2024-03-27T13:28:13ZengMDPI AGBiosensors2079-63742024-03-0114313910.3390/bios14030139A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper MembraneYanju Chen0Yuanyuan Zhu1Cheng Peng2Xiaofu Wang3Jian Wu4Huan Chen5Junfeng Xu6ZJU-Hangzhou Global Scientific and Technological Innovation Center, College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, ChinaZJU-Hangzhou Global Scientific and Technological Innovation Center, College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, ChinaKey Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, ChinaKey Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, ChinaZJU-Hangzhou Global Scientific and Technological Innovation Center, College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou 310058, ChinaHangzhou Digital-Micro Biotech Co., Ltd., Hangzhou 311215, ChinaKey Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, ChinaNucleic acid quantification, allowing us to accurately know the copy number of target nucleic acids, is significant for diagnosis, food safety, agricultural production, and environmental protection. However, current digital quantification methods require expensive instruments or complicated microfluidic chips, making it difficult to popularize in the point-of-care detection. Paper is an inexpensive and readily available material. In this study, we propose a simple and cost-effective paper membrane-based digital loop-mediated isothermal amplification (LAMP) method for nucleic acid quantification. In the presence of DNA fluorescence dyes, the high background signals will cover up the amplicons-formed bright spots. To reduce the background fluorescence signals, a quencher-fluorophore duplex was introduced in LAMP primers to replace non-specific fluorescence dyes. After that, the amplicons-formed spots on the paper membrane can be observed; thus, the target DNA can be quantified by counting the spots. Take <i>Vibrio parahaemolyticus</i> DNA detection as an instance, a good linear relationship is obtained between the light spots and the copy numbers of DNA. The paper membrane-based digital LAMP detection can detect 100 copies target DNA per reaction within 30 min. Overall, the proposed nucleic acid quantification method has the advantages of a simple workflow, short sample-in and answer-out time, low cost, and high signal-to-noise, which is promising for application in resourced limited areas.https://www.mdpi.com/2079-6374/14/3/139nucleic acid quantificationdigital detectionpaper membraneprobe-based LAMP |
| spellingShingle | Yanju Chen Yuanyuan Zhu Cheng Peng Xiaofu Wang Jian Wu Huan Chen Junfeng Xu A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane Biosensors nucleic acid quantification digital detection paper membrane probe-based LAMP |
| title | A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane |
| title_full | A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane |
| title_fullStr | A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane |
| title_full_unstemmed | A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane |
| title_short | A Point-of-Care Nucleic Acid Quantification Method by Counting Light Spots Formed by LAMP Amplicons on a Paper Membrane |
| title_sort | point of care nucleic acid quantification method by counting light spots formed by lamp amplicons on a paper membrane |
| topic | nucleic acid quantification digital detection paper membrane probe-based LAMP |
| url | https://www.mdpi.com/2079-6374/14/3/139 |
| work_keys_str_mv | AT yanjuchen apointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT yuanyuanzhu apointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT chengpeng apointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT xiaofuwang apointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT jianwu apointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT huanchen apointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT junfengxu apointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT yanjuchen pointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT yuanyuanzhu pointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT chengpeng pointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT xiaofuwang pointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT jianwu pointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT huanchen pointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane AT junfengxu pointofcarenucleicacidquantificationmethodbycountinglightspotsformedbylampampliconsonapapermembrane |