Sensitive methods for detection of SARS-CoV-2 RNA

Abstract

The occurrence of the COVID-19 pandemic caused by the SARS-CoV-2 virus since the end of 2019 has significantly affected the entire world. Now SARS-CoV-2 diagnostic tests are not only required for screening of suspected infected people for their medical treatment, but have also become a routine diagnosis for all people at a place where new cases have emerged in order to control spread of the disease from that region. For these reasons, sensitive methods for detection of SARS-CoV-2 are highly needed in order to avoid undetected infections. In addition, sample pooling that uses pooled specimens has been routinely employed as a time- and cost-effective strategy for community monitoring of SARS-CoV-2. In this regard, the content of each viral RNA sample of an individual will be further diluted in detection; therefore, higher detection sensitivity would be rather preferred. Among nucleic acid-based detection methods, isothermal nucleic acid amplifications are considered quite promising because they typically take less time to complete the test (even less than 20 min) without the need of thermal cycles. Hence, it does not necessitate the use of highly costly real-time PCR machines. According to recently published isothermal nucleic acid amplification methods, the reverse transcription recombinase polymerase amplification (RT-RPA) approach shows outstanding sensitivity with up to single-copy sensitivity in a test reaction. This chapter will mainly focus on how to employ RT-RPA technology to sensitively detect SARS-CoV-2 RNA. Besides, recently published RT-RPA based detection methods will be summarized and compared regarding their detection parameters and the primers and probes being used. In addition, we will also highlight the key considerations on how to design an ultrasensitive RT-RPA assay and the precautions needed to conduct the assay. Moreover, based on our recent report, we will also detail the methods we developed to detect SARS-CoV-2 RNA using modified RT-RPA, or RT-ERA, with single-copy sensitivity and the possible extensions beyond this method.

Keywords: Exo probe; Lateral flow; Nfo probe; RT-RPA; SARS-CoV-2 RNA; Single-copy sensitivity.

Fig. 1

Schematic illustration of the structure of coronavirus SARS-CoV-2 and its single-stranded RNA genome. The sequence information of the single-stranded RNA genome of SARS-CoV-2 serves as the basis for the development of nucleic acid-based diagnosis.

Fig. 2

Sensitive detection methods will be highly beneficial to prevent the spread of COVID-19. High sensitivity of diagnostic methods ensures low false negative results and therefore reduces the latent cross transmission of non-diagnosed but actually infected individuals to their closely contacted people.

Fig. 3

The general principle of recombinase polymerase amplification. Step I: recombinase and primer form complexes and target homologous DNA; step II: strand exchange forms a D-loop; herein, the brownish and blue-coloured arrows refer to the forward and reverse primers, respectively, upon annealing with their templates; step III: polymerase initiates synthesis; step IV: parental strands separate & synthesis continues; herein, the hollow boxed arrows refer to the directions of the polymerization reaction; step V: two duplexes form. Abbreviation(s): SSB, single-stranded DNA binding protein.

Fig. 4

Schematic view of the working principle of a typical exo probe for RT-RPA detection of viral RNA.

Fig. 5

Schematic view of the working mechanism of a nfo probe in RT-RPA for detection of viral RNA.

Fig. 6

Schematic view of the working mechanism of lateral flow detection (LFD) for the detection of bifunctional amplicon, i.e. amplicon with two antigenic labels, produced in RT-RPA reaction.

Fig. 7

The WEPEAR protocol seamlessly combines reverse transcription and RPA reactions in one tube allowing both steps to be conducted under their own optimal temperatures and prevents the Mg^2 + used in the RPA reaction from interacting with the RNA sample and inhibiting reverse transcription.