Abstract
Diagnosis of SARS-CoV-2 infection through rapid, accurate, and sensitive testing is the most important and fundamental step in coping with the COVID-19 epidemic. We have developed a sensitive fluorometric assay to detect SARS-CoV-2 viral RNA without thermal cycling. This assay system, based on tandem isothermal gene amplification (TIGA), is composed of ternary rolling circle amplification (t-RCA) and subsequent strand displacement amplification (SDA) coupled with G-quadruplex-generating RCA (SDA/GQ-RCA). Without the need to convert viral RNA into cDNA, viral RNA forms a ternary complex composed of hairpin primer (HP) and dumbbell padlock DNA during the t-RCA process. t-RCA generates a long chain of single-stranded DNA (ssDNA) with tandemly repeated hairpin structures that are subjected to SDA. SDA produces multiple short ssDNAs from t-RCA products, which then serve as primers for the second RCA reaction. A long ssDNA harboring repeated copies of the G-quadruplex is produced in the second round of RCA. Emission of enhanced fluorescence by thioflavin T, which intercalates into the G-quadruplex, allows fluorometric detection of amplified viral genes. This fluorometric analysis sensitively detected SARS-CoV-2 RNA as low as 5.9 aM, with a linear range between 0.2 fM and 200 fM within 1 h. Hence, this isothermal gene amplification method without reverse transcription of viral RNA can be applied to diagnose COVID-19 with high sensitivity and accuracy as an alternative to current PCR-based diagnosis.
Keywords: Isothermal gene amplification; Rolling circle amplification; SARS-CoV-2 viral RNA; Strand displacement amplification; Thioflavin T.
【저자키워드】 Rolling circle amplification, Thioflavin T., Isothermal gene amplification, SARS-CoV-2 viral RNA, Strand displacement amplification, 【초록키워드】 COVID-19, SARS-COV-2 infection, G-quadruplex, Diagnosis, amplification, DNA, sensitivity, Accuracy, reverse transcription, SARS-CoV-2 RNA, Viral RNA, diagnose, Rolling circle amplification, COVID-19 epidemic, Analysis, SARS-CoV-2 viral RNA, isothermal, cDNA, gene amplification, alternative, complex, primer, emission, Primers, viral genes, Thioflavin, single-stranded DNA, ssDNA, hairpin structure, Cycling, repeated, RCA, strand, SARS-CoV-2 viral, produced, amplified, detect, composed, subsequent, generate, form, linear, Rolling, 【제목키워드】 detection, amplification, isothermal,