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

https://doi.org/10.1016/j.bios.2021.113689Get rights and content

Highlights

  • The NESBA could detect SARS-CoV-2 gRNA down to 0.5 copies/μL for both E and N genes within 30 min under isothermal condition.

  • This strategy was able to identify a large cohort of clinical samples (n=98) with 100% clinical sensitivity and specificity.

  • This method was demonstrated multiplex capability, detecting the E and N genes of SARS-CoV-2 gRNA simultaneously in one-pot.

  • This work could serve as a powerful diagnostic platform to realize the point-of-care (POC) diagnosis for COVID-19.

Abstract

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.

Keywords

COVID-19
SARS-CoV-2
qRT-PCR
Isothermal amplification
NESBA
NASBA

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1

These authors equally contributed to this work.

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