Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing
Moraxella catarrhalis (M. catarrhalis) was an important pathogen closely associated with respiratory tract infections. We employed the loop-mediated isothermal amplification (LAMP) coupled with nanoparticle-based lateral flow biosensor (LFB) and fluorescence testing technique for formulating two dia...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Frontiers Media S.A.
2024-01-01
|
| Series: | Frontiers in Bioengineering and Biotechnology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1330047/full |
| _version_ | 1797362403090890752 |
|---|---|
| author | Fei Xiao Juan Zhou Xiaolan Huang Jin Fu Nan Jia Chunrong Sun Zheng Xu Yi Wang Lei Yu Lihui Meng |
| author_facet | Fei Xiao Juan Zhou Xiaolan Huang Jin Fu Nan Jia Chunrong Sun Zheng Xu Yi Wang Lei Yu Lihui Meng |
| author_sort | Fei Xiao |
| collection | DOAJ |
| description | Moraxella catarrhalis (M. catarrhalis) was an important pathogen closely associated with respiratory tract infections. We employed the loop-mediated isothermal amplification (LAMP) coupled with nanoparticle-based lateral flow biosensor (LFB) and fluorescence testing technique for formulating two diagnostic methods for M. catarrhalis detection, termed M. catarrhalis-LAMP-LFB assay and M. catarrhalis-LAMP-FRT, respectively. The M. catarrhalis-LAMP-LFB system incorporated the use of biotin-14-dCTP and a forward loop primer (LF) with a hapten at the 5′ end. This design in LAMP reaction enabled the production of double-labeled products that could be effectively analyzed using the lateral flow biosensor (LFB). For the M. catarrhalis-LAMP-FRT assay, the LF was modified with a sequence at 5′ end, and a fluorophore, as well as a black hole quencher, were strategically labeled at the 5′ end and within the middle of the new LF. The restriction endonuclease Nb.BsrDI could accurately recognize and cleave the newly synthesized double-strand terminal sequences, resulting in the separation of the fluorophore from the black hole quencher and releasing fluorescence signals. Both assays have been proven to be highly sensitive and specific, capable of detecting genomic DNA of M. catarrhalis at concentrations as low as 70 fg, with no cross-reactivity observed with non-M. catarrhalis pathogens. Furthermore, both methods successfully identified M. catarrhalis in all clinical samples within 1 h that were confirmed positive by real-time PCR, exhibiting superior sensitivity than conventional culture methods. Herein, the newly developed two LAMP-based assays were rapid and reliable for M. catarrhalis detection and hold significant promise for deployment in point-of-care (POC) settings. |
| first_indexed | 2024-03-08T16:06:33Z |
| format | Article |
| id | doaj.art-8d18812944d346468b3b70d82b588d00 |
| institution | Directory Open Access Journal |
| issn | 2296-4185 |
| language | English |
| last_indexed | 2024-03-08T16:06:33Z |
| publishDate | 2024-01-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Bioengineering and Biotechnology |
| spelling | doaj.art-8d18812944d346468b3b70d82b588d002024-01-08T05:15:32ZengFrontiers Media S.A.Frontiers in Bioengineering and Biotechnology2296-41852024-01-011110.3389/fbioe.2023.13300471330047Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testingFei Xiao0Juan Zhou1Xiaolan Huang2Jin Fu3Nan Jia4Chunrong Sun5Zheng Xu6Yi Wang7Lei Yu8Lihui Meng9Experiment Center, Capital Institute of Pediatrics, Beijing, ChinaExperiment Center, Capital Institute of Pediatrics, Beijing, ChinaExperiment Center, Capital Institute of Pediatrics, Beijing, ChinaExperiment Center, Capital Institute of Pediatrics, Beijing, ChinaExperiment Center, Capital Institute of Pediatrics, Beijing, ChinaExperiment Center, Capital Institute of Pediatrics, Beijing, ChinaExperiment Center, Capital Institute of Pediatrics, Beijing, ChinaExperiment Center, Capital Institute of Pediatrics, Beijing, ChinaDepartment of Infection Management, Children’s Hospital Affiliated to Capital Institute of Pediatrics, Beijing, ChinaDepartment of Infectious Diseases, Children’s Hospital Affiliated to Capital Institute of Pediatrics, Beijing, ChinaMoraxella catarrhalis (M. catarrhalis) was an important pathogen closely associated with respiratory tract infections. We employed the loop-mediated isothermal amplification (LAMP) coupled with nanoparticle-based lateral flow biosensor (LFB) and fluorescence testing technique for formulating two diagnostic methods for M. catarrhalis detection, termed M. catarrhalis-LAMP-LFB assay and M. catarrhalis-LAMP-FRT, respectively. The M. catarrhalis-LAMP-LFB system incorporated the use of biotin-14-dCTP and a forward loop primer (LF) with a hapten at the 5′ end. This design in LAMP reaction enabled the production of double-labeled products that could be effectively analyzed using the lateral flow biosensor (LFB). For the M. catarrhalis-LAMP-FRT assay, the LF was modified with a sequence at 5′ end, and a fluorophore, as well as a black hole quencher, were strategically labeled at the 5′ end and within the middle of the new LF. The restriction endonuclease Nb.BsrDI could accurately recognize and cleave the newly synthesized double-strand terminal sequences, resulting in the separation of the fluorophore from the black hole quencher and releasing fluorescence signals. Both assays have been proven to be highly sensitive and specific, capable of detecting genomic DNA of M. catarrhalis at concentrations as low as 70 fg, with no cross-reactivity observed with non-M. catarrhalis pathogens. Furthermore, both methods successfully identified M. catarrhalis in all clinical samples within 1 h that were confirmed positive by real-time PCR, exhibiting superior sensitivity than conventional culture methods. Herein, the newly developed two LAMP-based assays were rapid and reliable for M. catarrhalis detection and hold significant promise for deployment in point-of-care (POC) settings.https://www.frontiersin.org/articles/10.3389/fbioe.2023.1330047/fullMoraxella catarrhalisloop-mediated isothermal amplificationnanoparticle-based lateral flow biosensorrestriction endonucleasevisual detection |
| spellingShingle | Fei Xiao Juan Zhou Xiaolan Huang Jin Fu Nan Jia Chunrong Sun Zheng Xu Yi Wang Lei Yu Lihui Meng Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing Frontiers in Bioengineering and Biotechnology Moraxella catarrhalis loop-mediated isothermal amplification nanoparticle-based lateral flow biosensor restriction endonuclease visual detection |
| title | Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing |
| title_full | Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing |
| title_fullStr | Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing |
| title_full_unstemmed | Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing |
| title_short | Rapid and reliable diagnosis of Moraxella catarrhalis infection using loop-mediated isothermal amplification-based testing |
| title_sort | rapid and reliable diagnosis of moraxella catarrhalis infection using loop mediated isothermal amplification based testing |
| topic | Moraxella catarrhalis loop-mediated isothermal amplification nanoparticle-based lateral flow biosensor restriction endonuclease visual detection |
| url | https://www.frontiersin.org/articles/10.3389/fbioe.2023.1330047/full |
| work_keys_str_mv | AT feixiao rapidandreliablediagnosisofmoraxellacatarrhalisinfectionusingloopmediatedisothermalamplificationbasedtesting AT juanzhou rapidandreliablediagnosisofmoraxellacatarrhalisinfectionusingloopmediatedisothermalamplificationbasedtesting AT xiaolanhuang rapidandreliablediagnosisofmoraxellacatarrhalisinfectionusingloopmediatedisothermalamplificationbasedtesting AT jinfu rapidandreliablediagnosisofmoraxellacatarrhalisinfectionusingloopmediatedisothermalamplificationbasedtesting AT nanjia rapidandreliablediagnosisofmoraxellacatarrhalisinfectionusingloopmediatedisothermalamplificationbasedtesting AT chunrongsun rapidandreliablediagnosisofmoraxellacatarrhalisinfectionusingloopmediatedisothermalamplificationbasedtesting AT zhengxu rapidandreliablediagnosisofmoraxellacatarrhalisinfectionusingloopmediatedisothermalamplificationbasedtesting AT yiwang rapidandreliablediagnosisofmoraxellacatarrhalisinfectionusingloopmediatedisothermalamplificationbasedtesting AT leiyu rapidandreliablediagnosisofmoraxellacatarrhalisinfectionusingloopmediatedisothermalamplificationbasedtesting AT lihuimeng rapidandreliablediagnosisofmoraxellacatarrhalisinfectionusingloopmediatedisothermalamplificationbasedtesting |