We herein describe rapid and accurate clinical testing for COVID-19 by nicking and extension chain reaction system-based amplification (NESBA), an ultrasensitive version of NASBA. The primers to identify SARS-CoV-2 viral RNA were designed to additionally contain the nicking recognition sequence at the 5′-end of conventional NASBA primers, which would enable nicking enzyme-aided exponential amplification of T7 RNA promoter-containing double-stranded DNA (T7DNA). As a consequence of this substantially enhanced amplification power, the NESBA technique was able to ultrasensitively detect SARS-CoV-2 genomic RNA (gRNA) down to 0.5 copies/μL (= 10 copies/reaction) for both envelope (E) and nucleocapsid (N) genes within 30 min under isothermal temperature (41 °C). When the NESBA was applied to test a large cohort of clinical samples (n = 98), the results fully agreed with those from qRT-PCR and showed the excellent accuracy by yielding 100% clinical sensitivity and specificity. By employing multiple molecular beacons with different fluorophore labels, the NESBA was further modulated to achieve multiplex molecular diagnostics, so that the E and N genes of SARS-CoV-2 gRNA were simultaneously assayed in one-pot. By offering the superior analytical performances over the current qRT-PCR, the isothermal NESBA technique could serve as very powerful platform technology to realize the point-of-care (POC) diagnosis for COVID-19.
|Journal||Biosensors and Bioelectronics|
|Publication status||Published - 2022 Jan 15|
Bibliographical noteFunding Information:
This research was supported by BioNano Health-Guard Research Center funded by the Ministry of Science and ICT (MSIT) of Korea as Global Frontier Project (Grant number H-GUARD_2013M3A6B2078964) and Kyung Nam Pharm Co.Ltd.
This research was supported by BioNano Health-Guard Research Center funded by the Ministry of Science and ICT ( MSIT ) of Korea as Global Frontier Project (Grant number H-GUARD_2013M3A6B2078964 ) and Kyung Nam Pharm Co.,Ltd.
© 2021 Elsevier B.V.
All Science Journal Classification (ASJC) codes
- Biomedical Engineering