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Isothermal molecular diagnostic strategy for rapid and on-site detection of SARS-CoV-2 and emerging variants
Writer 김지애
Date 2022-09-20 10:57:28.0
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Isothermal molecular diagnostic strategy for rapid and on-site detection of SARS-CoV-2 and emerging variants

   

Korean researcher in Konkuk University developed a simple and rapid method, termed as the split T7 promoter-based isothermal transcription amplification with light-up RNA aptamer (STAR), for one-pot, multiplex detection of SARS-CoV-2 and emerging variants. The study appears in the journal of Biosensors and Bioelectronics in 2022, July. 

   

In December 2019, an infectious respiratory RNA virus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged and rapidly spread around the world. As of March 2022, over 487 million people have been infected and 6.1 million have died. Additionally, variants of SARS-CoV-2 with the 23,403 A > G mutation (p. D614G), conferring greater infectivity and more rapid spread, have become predominant in various regions. Therefore, there is an urgent need for rapid detection of SARS-CoV-2 to isolate infected people in a timely manner and prevent further viral transmission

   

The researchers developed a new isothermal nucleic acid amplification technology that can quickly detect SARS-CoV-2 while addressing the limitations of previous molecular diagnostic strategies. STAR is a three-way junction-based technology that can detect target RNA with one enzyme in a single step. Through systematic investigation of the T7 promoter, the researchers found that the transcription reaction effectively occurs with the split T7 promoter at a ratio of 4:16. This feature was combined with the three-way junction structure and light-up RNA aptamers to achive detection of the SARS-CoV-2 N gene in less than 30 min at 37 °C without the need for initial denaturation and cooling steps. The practical applicability of the proposed strategy was evaluated by determining its detection performance in 60 clinical samples. Moreover, STAR was utilized for multiplex detection of the D614G mutation and N gene of SARS-CoV-2 in a single tube as well as for the direct detection of bacterial 16S rRNA without additional nucleic acid purification, thereby confirming the wide applicability of this method for nucleic acid biomarker detection.

   

Prof. Park said that “The proposed strategy requires only one enzyme (T7 RNA polymerase), thereby allowing for the one-step, rapid detection (30 min, 37 °C) of target RNA biomarkers. It could be utilized in a variety of molecular biology applications as well as for precision diagnostics. We believe that our study will make a significant and practical impact on the society because, considering the coronavirus disease (COVID-19) pandemic, there is an urgent need for rapid viral RNA detection methods to be used in the clinical setting without the need for extensive laboratory equipment”.

   

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Schematic illustration of split T7 promoter-based isothermal transcription amplification with light-up RNA aptamers (STAR) for one-pot, multiplex detection of SARS-CoV-2 and emerging variants. In the presence of the target RNA, STAR DNA probes can form a three-way junction structure and complete the double-stranded T7 promoter with a nick site. Thus, T7 RNA polymerase can bind to the T7 promoter and initiate transcription for the generation of a large amount of light-up RNA aptamers. Subsequently, the light-up RNA aptamers combine with the fluorogenic dye, resulting in the generation of a highly enhanced fluorescent signal. The one-step reaction occurs at 37 °C within 30 min.

   

[Reference] Taehwi Yoon, Jiye Shin, Hyun-Jung Choi, and Ki Soo Park, "Split T7 promoter-based isothermal transcription amplification for one-step fluroescence detection of SARS-CoV-2 and emerging variants", Biosensors and Bioelectronics, doi.org/10.1016/j.bios.2022.114221

   

[Main Author] Taehwi Yoon(Konkuk University), Jiye Shin(Konkuk University), Ki Soo Park(Konkuk University) 

* Contact : Professor Ki Soo Park (kskonkuk@gmail.com or akdong486@konkuk.ac.kr)