Rapid detection of SARS-CoV-2: The gradual boom of lateral flow immunoassay

Abstract

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is still in an epidemic situation, which poses a serious threat to the safety of people and property. Rapid diagnosis and isolation of infected individuals are one of the important methods to control virus transmission. Existing lateral flow immunoassay techniques have the advantages of rapid, sensitive, and easy operation, and some new options have emerged with the continuous development of nanotechnology. Such as lateral flow immunoassay test strips based on colorimetric-fluorescent dual-mode and gold nanoparticles, Surface Enhanced Raman Scattering, etc., these technologies have played an important role in the rapid diagnosis of COVID-19. In this paper, we summarize the current research progress of lateral flow immunoassay in the field of Severe Acute Respiratory Syndrome Coronavirus 2 infection diagnosis, analyze the performance of Severe Acute Respiratory Syndrome Coronavirus 2 lateral flow immunoassay products, review the advantages and limitations of different detection methods and markers, and then explore the competitive CRISPR-based nucleic acid chromatography detection method. This method combines the advantages of gene editing and lateral flow immunoassay and can achieve rapid and highly sensitive lateral flow immunoassay detection of target nucleic acids, which is expected to be the most representative method for community and clinical point-of-care testing. We hope that researchers will be inspired by this review and strive to solve the problems in the design of highly sensitive targets, the selection of detection methods, and the enhancement of CRISPR technology, to truly achieve rapid, sensitive, convenient, and specific detection of novel coronaviruses, thus promoting the development of novel coronavirus diagnosis and contributing our modest contribution to the world's fight against epidemics.

Keywords: COVID-19; CRISPR; SARS-CoV-2; antibody; antigen; lateral flow immunoassay; nanotechnology; nucleic acid.

FIGURE 1

Structure of SARS-CoV-2. Illustrative scheme: This figure shows the approximate protein structure of SARS-CoV-2 and the gene fragment of RNA, in addition to depicting the way S1 binds to the ACE2 receptor to invade the organism. As can be seen in the figure, except for the N protein which is wrapped around the nucleic acid, the E, M, and S proteins are anchored to the envelope, each in its way. Among them, the S protein consists of two subunits, S1 and S2. S1 protein has a receptor binding domain (RBD) on it that binds to ACE2 on the host cell. S2 protein has a more complex structure and its role is to fuse the virus to the host cell membrane. In the pre-fusion conformation, the S1 and S2 subunits remain non-covalently bound. When the novel coronavirus wants to enter the host cell, the S protein shifts from the closed to the open state, thus mediating entry into the host cell (Lan et al., 2020; Shang et al., 2020; Walls et al., 2020). In addition, when RBD is structured and ACE2, it is activated by proteases such as TMPRSS2 on the host cell membrane, which causes the S1/S2 enzymatic cleavage site to be cut, thus facilitating the fusion of the virus with the host cell membrane (Zhou et al., 2020). The genetic structure of the virus mentioned in the paper is also briefly shown in Fig. The RNA genome of SARS-CoV-2 consists of 14 open reading frames (ORFs) (Khailany et al., 2020). Where ORF1a and ORF1b overlap at the (-1) ribosomal frameshift, this part contains about two-thirds of the genome and is mainly used for processing and synthesis of non-borrowed proteins. The remaining one-third of the genome in which the different ORFs also overlap each other is mainly used to encode four structural proteins (Arya et al., 2021). (This figure created with BioRender.com).

FIGURE 2

The CRISPR-Cas system for the whole process of COVID-19 immunochromatography detection. Illustrative scheme:The figure briefly summarizes the technical flow of most current CRISPR combined nucleic acid amplification and LFIA using the method designed by Zhu et al. (2021) (A) In total, there are three major steps before using immunochromatographic test strips. First, a nasal or pharyngeal swab is used to collect a sample from the subject and extract RNA. second, the nucleic acid is amplified, and the reverse transcription amplification method is generally chosen, in which the extracted RNA is specifically amplified after binding to a probe modified with a fluorescent moiety (fluorescein) and a quenching moiety (biotin), during which the target RNA is converted into a target for more DNA. Then the CRISPR-Cas system is applied to cut the target gene motif, but because Cas12 and Cas13 cut RNA and DNA respectively, the nucleic acid also needs to be transcribed to RNA when Cas13 is selected. Third, the CRISPR-Cas system cuts and a probe with the target RNA or DNA bound to the Cas protein in advance is used to identify the target nucleic acid The CRISPR-Cas system is activated when the target nucleic acid, which has been modified by fluorescent and quenching motifs, binds to the probe and cleaves the nucleic acid sequence non-selectively. The final target nucleic acid sequence is cleaved and the fluorophore fluoresces, allowing the detection of the signal (Ramachandran and Santiago, 2021). (B) The test strip located at the top is a schematic diagram of the structure of the product, and the one at the bottom is presented after receiving a positive sample. The above processed products were transferred to LFIA strips for examination, and the cleaved and uncleaved nucleic acid fragments were first labeled with colloidal gold and then flowed to detection and quality control lines immobilized with anti-biotin and anti-luciferin antibodies, respectively, so that the aggregation of uncleaved nucleic acid fragments could show a red band, while the cleaved nucleic acid fragments could be detected by a fluorescence detector. (This figure created with BioRender.com).