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Multiplex reverse transcription loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for the diagnosis of COVID-19.
Biosens Bioelectron. 2020 Oct 15; 166:112437.BB

Abstract

The ongoing global pandemic (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a huge public health issue. Hence, we devised a multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (mRT-LAMP-LFB) for diagnosing COVID-19. Using two LAMP primer sets, the ORF1ab (opening reading frame 1a/b) and N (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified in a single-tube reaction, and detected with the diagnosis results easily interpreted by LFB. In presence of FITC (fluorescein)-/digoxin- and biotin-labeled primers, mRT-LAMP produced numerous FITC-/digoxin- and biotin-attached duplex amplicons, which were determined by LFB through immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the test line of LFB) and biotin/treptavidin interaction (biotin on the duplex and strptavidin on the polymerase nanoparticle). The accumulation of nanoparticles leaded a characteristic crimson band, enabling multiplex analysis of ORF1ab and N gene without instrumentation. The limit of detection (LoD) of COVID-19 mRT-LAMP-LFB was 12 copies (for each detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. The analytical sensitivity of SARS-CoV-2 was 100% (33/33 oropharynx swab samples collected from COVID-19 patients), and the assay's specificity was also 100% (96/96 oropharynx swab samples collected from non-COVID-19 patients). The total diagnostic test can be completed within 1 h from sample collection to result interpretation. In sum, the COVID-19 mRT-LAMP-LFB assay is a promising tool for diagnosing SARS-CoV-2 infections in frontline public health field and clinical laboratories, especially from resource-poor regions.

Authors+Show Affiliations

Central & Clinical Laboratory of Sanya People's Hospital, Sanya, Hainan, 572000, PR China.Central & Clinical Laboratory of Sanya People's Hospital, Sanya, Hainan, 572000, PR China.Central & Clinical Laboratory of Sanya People's Hospital, Sanya, Hainan, 572000, PR China.Central & Clinical Laboratory of Sanya People's Hospital, Sanya, Hainan, 572000, PR China.Central & Clinical Laboratory of Sanya People's Hospital, Sanya, Hainan, 572000, PR China.Central & Clinical Laboratory of Sanya People's Hospital, Sanya, Hainan, 572000, PR China.Central & Clinical Laboratory of Sanya People's Hospital, Sanya, Hainan, 572000, PR China.Central & Clinical Laboratory of Sanya People's Hospital, Sanya, Hainan, 572000, PR China.Central & Clinical Laboratory of Sanya People's Hospital, Sanya, Hainan, 572000, PR China.Central & Clinical Laboratory of Sanya People's Hospital, Sanya, Hainan, 572000, PR China.Wenchang People's Hospital, Wenchang, Hainan, 572000, PR China.Central & Clinical Laboratory of Sanya People's Hospital, Sanya, Hainan, 572000, PR China.Department of Respiratory Disease, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing, 10045, PR China; Key Laboratory of Major Diseases in Children, Ministry of Education, Beijing Key Laboratory of Pediatric Respiratory Infection Disease, National Clinical Research Center for Respiratory Diseases, Beijing Children's Hospital, Capital Medical University, National Center for Children's Health, Beijing, 10045, PR China. Electronic address: wildwolf0101@163.com.

Pub Type(s)

Journal Article
Validation Study

Language

eng

PubMed ID

32692666

Citation

Zhu, Xiong, et al. "Multiplex Reverse Transcription Loop-mediated Isothermal Amplification Combined With Nanoparticle-based Lateral Flow Biosensor for the Diagnosis of COVID-19." Biosensors & Bioelectronics, vol. 166, 2020, p. 112437.
Zhu X, Wang X, Han L, et al. Multiplex reverse transcription loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for the diagnosis of COVID-19. Biosens Bioelectron. 2020;166:112437.
Zhu, X., Wang, X., Han, L., Chen, T., Wang, L., Li, H., Li, S., He, L., Fu, X., Chen, S., Xing, M., Chen, H., & Wang, Y. (2020). Multiplex reverse transcription loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for the diagnosis of COVID-19. Biosensors & Bioelectronics, 166, 112437. https://doi.org/10.1016/j.bios.2020.112437
Zhu X, et al. Multiplex Reverse Transcription Loop-mediated Isothermal Amplification Combined With Nanoparticle-based Lateral Flow Biosensor for the Diagnosis of COVID-19. Biosens Bioelectron. 2020 Oct 15;166:112437. PubMed PMID: 32692666.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Multiplex reverse transcription loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for the diagnosis of COVID-19. AU - Zhu,Xiong, AU - Wang,Xiaoxia, AU - Han,Limei, AU - Chen,Ting, AU - Wang,Licheng, AU - Li,Huan, AU - Li,Sha, AU - He,Lvfen, AU - Fu,Xiaoying, AU - Chen,Shaojin, AU - Xing,Mei, AU - Chen,Hai, AU - Wang,Yi, Y1 - 2020/07/15/ PY - 2020/05/15/received PY - 2020/06/13/revised PY - 2020/07/07/accepted PY - 2020/7/22/pubmed PY - 2020/9/4/medline PY - 2020/7/22/entrez KW - COVID-19 KW - Lateral flow biosensor KW - Multiplex reverse transcription loop-mediated isothermal amplification KW - Rapid diagnosis KW - SARS-CoV-2 SP - 112437 EP - 112437 JF - Biosensors & bioelectronics JO - Biosens Bioelectron VL - 166 N2 - The ongoing global pandemic (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a huge public health issue. Hence, we devised a multiplex reverse transcription loop-mediated isothermal amplification (mRT-LAMP) coupled with a nanoparticle-based lateral flow biosensor (LFB) assay (mRT-LAMP-LFB) for diagnosing COVID-19. Using two LAMP primer sets, the ORF1ab (opening reading frame 1a/b) and N (nucleoprotein) genes of SARS-CoV-2 were simultaneously amplified in a single-tube reaction, and detected with the diagnosis results easily interpreted by LFB. In presence of FITC (fluorescein)-/digoxin- and biotin-labeled primers, mRT-LAMP produced numerous FITC-/digoxin- and biotin-attached duplex amplicons, which were determined by LFB through immunoreactions (FITC/digoxin on the duplex and anti-FITC/digoxin on the test line of LFB) and biotin/treptavidin interaction (biotin on the duplex and strptavidin on the polymerase nanoparticle). The accumulation of nanoparticles leaded a characteristic crimson band, enabling multiplex analysis of ORF1ab and N gene without instrumentation. The limit of detection (LoD) of COVID-19 mRT-LAMP-LFB was 12 copies (for each detection target) per reaction, and no cross-reactivity was generated from non-SARS-CoV-2 templates. The analytical sensitivity of SARS-CoV-2 was 100% (33/33 oropharynx swab samples collected from COVID-19 patients), and the assay's specificity was also 100% (96/96 oropharynx swab samples collected from non-COVID-19 patients). The total diagnostic test can be completed within 1 h from sample collection to result interpretation. In sum, the COVID-19 mRT-LAMP-LFB assay is a promising tool for diagnosing SARS-CoV-2 infections in frontline public health field and clinical laboratories, especially from resource-poor regions. SN - 1873-4235 UR - https://www.unboundmedicine.com/medline/citation/32692666/Multiplex_reverse_transcription_loop_mediated_isothermal_amplification_combined_with_nanoparticle_based_lateral_flow_biosensor_for_the_diagnosis_of_COVID_19_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0956-5663(20)30431-0 DB - PRIME DP - Unbound Medicine ER -