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Duplex Reverse Transcription Multienzyme Isothermal Rapid Amplification Assays for Detecting SARS-CoV-2 by Meiyun Shang, Jin Li, Xiange Sun, Ning Su, Bowen Li, Yuansu Jiang, Huimin Chong, Weiping Lu, Qing Huang

Background: Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that brings a significant public health challenge. A rapid and simple method is necessary for testing suspected samples and screening the population.
Methods: To better monitor sample effectiveness, this study described a method to detect nucleocapsid protein gene (N gene) of SARS-CoV-2 and human ACTB gene employing real-time duplex reverse transcription multienzyme isothermal rapid amplification (RT-MIRA) assays.
Results: The established real-time duplex RT-MIRA assays showed that no cross-reactions were observed to other pathogens and the detection limit was 100 copies/reaction. Using simulated clinical samples to test established assays further and the amplification process took no more than 20 minutes at 42°C.
Conclusions: RT-MIRA assays are faster and easier than reverse transcription real-time polymerase chain reaction (RT-PCR). It is expected to be further optimized and evaluated in the detection of SARS-CoV-2 confirmed cases.

DOI: 10.7754/Clin.Lab.2021.210239