Split T7 promoter-based isothermal transcription amplification for one-step fluorescence detection of SARS-CoV-2 and emerging variants

• Published in: Biosensors & bioelectronics Vol. 208; p. 114221
• Main Authors: Yoon, Taehwi, Shin, Jiye, Choi, Hyun-Jung, Park, Ki Soo
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 15.07.2022
• Subjects: Aptamers, Nucleotide; Biosensing Techniques; COVID-19 - diagnosis; Humans; Isothermal amplification; Light-up RNA aptamer; Molecular Diagnostic Techniques - methods; Nucleic Acid Amplification Techniques - methods; RNA, Ribosomal, 16S; RNA, Viral - genetics; SARS-CoV-2; SARS-CoV-2 - genetics; Sensitivity and Specificity; Split T7 promoter; Three-way junction; Transcription; Split T7 promoter; SARS-CoV-2; Transcription; Isothermal amplification; Three-way junction; Light-up RNA aptamer
• ISSN: 0956-5663; 1873-4235; 1873-4235
• DOI: 10.1016/j.bios.2022.114221

The negative global impact of the coronavirus disease pandemic has highlighted the crucial need for a rapid and convenient method of viral RNA detection. In this study, we report a novel method, termed as the split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR), for one-pot detection of viral RNA. STAR uses a split T7 promoter that is applied to a three-way junction to mediate the selective transcription by the T7 RNA polymerase in the presence of target RNA. In addition, a light-up RNA aptamer is used for signal amplification. STAR can detect viral RNA in less than 30 min with high specificity and sensitivity. By testing of 60 nasopharyngeal SARS-CoV-2 samples, the STAR assay demonstrates an excellent sensitivity and specificity of 96.7% and 100%, respectively. Moreover, we provide experimental evidence of the broad applicability of this assay through the multiplex detection of SARS-CoV-2 variants (D614G mutation) and direct detection of bacterial 16S rRNA.