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; Isothermal amplification; NASBA; NESBA; SARS-CoV-2; qRT-PCR.
【저자키워드】 COVID-19, SARS-CoV-2, isothermal amplification, NASBA, NESBA, qRT-PCR., 【초록키워드】 qRT-PCR, POC, Diagnosis, diagnostics, RNA, point-of-care, amplification, clinical samples, DNA, specificity, Viral, Accuracy, nucleocapsid, temperature, large cohort, isothermal amplification, molecular, N gene, multiplex, platform, NASBA, SARS-CoV-2 viral RNA, genomic RNA, isothermal, clinical sensitivity, Chain Reaction, Double-stranded DNA, sequence, primer, clinical testing, extension, Primers, gRNA, clinical sample, identify, detect, applied, double-stranded, modulated, yielding, 【제목키워드】 amplification, Rapid, clinical testing, extension,