A Ligation/Recombinase Polymerase Amplification Assay for Rapid Detection of SARS-CoV−2

Frontiers in Cellular and Infection Microbiology

Chao Ma¹  Song Gao¹  Lizhan Chen¹  Xue Zhang¹  Yang Cao¹  Longyu Yi²  Pei Wang²  Xin Liu²  Qunwei Lu² 

[1] Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Key Laboratory of Marine Biological Resources and Environment, Co-Innovation Center of Jiangsu Marine Bio-industry Technology, School of Pharmacy, Jiangsu Ocean University, Lianyungang, China;Key Laboratory of Molecular Biophysics of Ministry of Education, Department of Biomedical Engineering, College of Life Science and Technology, Center for Human Genome Research, Huazhong University of Science and Technology, Wuhan, China;

关键词: SARS-CoV-2;    T4 DNA ligase;    ligation;    recombinase polymerase amplification;    nucleic acid detection;   

DOI  :  10.3389/fcimb.2021.680728

来源: Frontiers

【 摘 要 】

The pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has led to more than 117 million reported cases and 2.6 million deaths. Accurate diagnosis technologies are vital for controlling this pandemic. Reverse transcription (RT)-based nucleic acid detection assays have been developed, but the strict sample processing requirement of RT has posed obstacles on wider applications. This study established a ligation and recombinase polymerase amplification (L/RPA) combined assay for rapid detection of SARS-CoV−2 on genes N and ORF1ab targeting the specific biomarkers recommended by the China CDC. Ligase-based strategies usually have a low-efficiency problem on RNA templates. This study has addressed this problem by using a high concentration of the T4 DNA ligase and exploiting the high sensitivity of RPA. Through selection of the ligation probes and optimization of the RPA primers, the assay achieved a satisfactory sensitivity of 101 viral RNA copies per reaction, which was comparable to RT-quantitative polymerase chain reaction (RT-qPCR) and other nucleic acid detection assays for SARS-CoV−2. The assay could be finished in less than 30 min with a simple procedure, in which the requirement for sophisticated thermocycling equipment had been avoided. In addition, it avoided the RT procedure and could potentially ease the requirement for sample processing. Once validated with clinical samples, the L/RPA assay would increase the practical testing availability of SARS-CoV-2. Moreover, the principle of L/RPA has an application potential to the identification of concerned mutations of the virus.

【 授权许可】

CC BY