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

Abstract

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.

A DNA–Cu nanocluster and exonuclease I integrated label-free reporting system for CRISPR/Cas12a-based SARS-CoV-2 detection with minimized background signals

“…To further prove the practicability of CRISPR-CNS, we purchased the SARS-CoV-2 RNA standard from the Chinese Academy of Metrology as the test sample (Table 2). 62,63 The SARS-CoV-2 RNA standard contained 10 5 copies per μL N gene, 10 5 copies per μL E gene, and 10 6 copies per μL ORF1ab gene fragments (sequences detailed in Table S1, ESI†). This SARS-CoV-2 RNA standard sample was diluted into different concentration gradients of the N gene from 10 copies per μL to 10 5 copies per μL.…”

A precise review on NAATs‐based diagnostic assays for COVID‐19: A motion in fast POC molecular tests

“…The performance of the assay relies on the viral RNA template, T4 DNA ligase and ligation probes set including probe A and probe B. 30 Notwithstanding the inefficiency of DNA ligases in RNA templates, this assay resolved the problem by optimizing the ligation protocol and employing the high sensitivity of RPA. 31 , 32 Each probe consists of a part annealing to the RNA and an ‘amplification arm’ to promote the RPA amplification.…”

Current trends in COVID-19 diagnosis and its new variants in physiological fluids: Surface antigens, antibodies, nucleic acids, and RNA sequencing

“…However, ligase-based strategies usually have the problem of low efficiency in RNA templates. Therefore, Wang and co-workers [ 28 ] developed a L/RPA bioassay for the rapid detection of SARS-CoV-2 on the N and ORF1ab genes targeting the specific biomarkers. The authors overcame the problem of the low efficiency of the RNA template method by using a high concentration of T4 DNA ligase and taking advantage of the high sensitivity of recombinase polymerase amplification.…”

“…The review is structured according to the target molecule (e.g., the whole virus or its antigenic proteins, the host antibody, and the viral gene). A further subdivision is based on the different methods of detection, including lateral flow immunoassay (LFIA) [ 22 , 23 ], enzyme-linked immunosorbent assay (ELISA) [ 24 ], biosensors (optical, electrochemical, and electronics) [ 25 ], reverse transcription-polymerase chain reaction (RT-PCR) [ 26 , 27 ], recombinase polymerase amplification (RPA) [ 28 ], reverse transcription loop-mediated isothermal amplification (RT-LAMP) [ 29 ], DNA microarray [ 30 ], and clusters of regularly interspaced short palindromic repeats [ 31 , 32 ]. We also discuss recent developments and challenges of SARS-CoV-2 detection techniques, such as early diagnosis of infection, limit of detection, analytical selectivity, and clinical specificity.…”