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Rapid and accurate clinical testing for COVID-19 by nicking and extension chain reaction system-based amplification (NESBA)

 Yong Ju  ;  Jaemin Kim  ;  Yeonkyung Park  ;  Chang Yeol Lee  ;  Kyungnam Kim  ;  Ki Ho Hong  ;  Hyukmin Lee  ;  Dongeun Yong  ;  Hyun Gyu Park 
 BIOSENSORS & BIOELECTRONICS, Vol.196 : 113689, 2022-01 
Journal Title
Issue Date
COVID-19 ; Isothermal amplification ; NASBA ; NESBA ; SARS-CoV-2 ; qRT-PCR
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.
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1. College of Medicine (의과대학) > Dept. of Laboratory Medicine (진단검사의학교실) > 1. Journal Papers
Yonsei Authors
Yong, Dong Eun(용동은) ORCID logo https://orcid.org/0000-0002-1225-8477
Lee, Hyuk Min(이혁민) ORCID logo https://orcid.org/0000-0002-8523-4126
Hong, Ki Ho(홍기호) ORCID logo https://orcid.org/0000-0002-5700-9036
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