Multiplex reverse transcription loop-mediated isothermal amplification combined with nanoparticle-based lateral flow biosensor for the diagnosis of COVID-19

被引:302
作者
Zhu, Xiong [1 ]
Wang, Xiaoxia [1 ]
Han, Limei [1 ]
Chen, Ting [1 ]
Wang, Licheng [1 ]
Li, Huan [1 ]
Li, Sha [1 ]
He, Lvfen [1 ]
Fu, Xiaoying [1 ]
Chen, Shaojin [1 ]
Xing, Mei [2 ]
Chen, Hai [1 ]
Wang, Yi [3 ,4 ]
机构
[1] Sanya Peoples Hosp, Cent & Clin Lab, Sanya 572000, Hainan, Peoples R China
[2] Wenchang Peoples Hosp, Wenchang 572000, Hainan, Peoples R China
[3] Capital Med Univ, Beijing Childrens Hosp, Natl Ctr Childrens Hlth, Dept Resp Dis,Beijing Pediat Res Inst, Beijing 10045, Peoples R China
[4] Capital Med Univ, Beijing Key Lab Pediat Resp Infect Dis, Natl Ctr Childrens Hlth,Natl Clin Res Ctr Resp Di, Key Lab Major Dis Children,Minist Educ,Beijing Ch, Beijing 10045, Peoples R China
关键词
SARS-CoV-2; COVID-19; Multiplex reverse transcription loop-mediated; isothermal amplification; Lateral flow biosensor; Rapid diagnosis;
D O I
10.1016/j.bios.2020.112437
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
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.
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页数:7
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