Advancements in detection of SARS-CoV-2 infection for confronting COVID-19 pandemics

Abstract

As one of the major approaches in combating the COVID-19 pandemics, the availability of specific and reliable assays for the SARS-CoV-2 viral genome and its proteins is essential to identify the infection in suspected populations, make diagnoses in symptomatic or asymptomatic individuals, and determine clearance of the virus after the infection. For these purposes, use of the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) for detection of the viral nucleic acid remains the most valuable in terms of its specificity, fast turn-around, high-throughput capacity, and reliability. It is critical to update the sequences of primers and probes to ensure the detection of newly emerged variants. Various assays for increased levels of IgG or IgM antibodies are available for detecting ongoing or past infection, vaccination responses, and persistence and for identifying high titers of neutralizing antibodies in recovered individuals. Viral genome sequencing is increasingly used for tracing infectious sources, monitoring mutations, and subtype classification and is less valuable in diagnosis because of its capacity and high cost. Nanopore target sequencing with portable options is available for a quick process for sequencing data. Emerging CRISPR-Cas-based assays, such as SHERLOCK and AIOD-CRISPR, for viral genome detection may offer options for prompt and point-of-care detection. Moreover, aptamer-based probes may be multifaceted for developing portable and high-throughput assays with fluorescent or chemiluminescent probes for viral proteins. In conclusion, assays are available for viral genome and protein detection, and the selection of specific assays depends on the purposes of prevention, diagnosis and pandemic control, or monitoring of vaccination efficacy.

Fig. 1

Illustration of the SARS-CoV-2 viral genome, proteins and corresponding assays. The genome of SARS-CoV-2 is a positive single-stranded RNA with more than 30,000 bp nucleotides. The capsid outside the genome is formed by the nucleocapsid protein (N) and is further wrapped by an envelope composed of three structural proteins: membrane protein (M), spike protein (S), and envelope protein (E). The entry of coronavirus into host cells is mediated by the S protein, which is a homotrimer protruding from the viral envelope that recognizes the receptor angiotensin-converting enzyme 2 (ACE2) via the S1 receptor-binding domain (RBD) and uses the S2 domain for fusion with the host cell membrane to enter host cells. In addition to these four structural proteins, SARS-CoV-2 contains sixteen nonstructural proteins (NSPs). Four NSPs responsible for viral replication or transcription are shown in this illustration. NSP3 separates the translated protein. NSP5 is responsible for cleaving the viral polyprotein into functional units during replication. NSP12 contains the RNA-dependent RNA polymerase (RdRp). NSP13 participates in viral replication or transcription via the zinc-binding domain. ACE2 angiotensin-converting enzyme 2, E envelope protein, M transmembrane protein, N nucleocapsid protein, NSP nonstructural protein, ORF open reading frame, RdRp RNA-dependent RNA polymerase, S spike protein.

Fig. 2

Viral open reading frames (ORFs) and mutations in primer or probe regions. A Four open reading frames (ORFs) in the viral genome are indicated for encoding viral structural or nonstructural proteins. B–E Mutations have been found in primer and probe regions in various countries and regions. As indicated, most mutations occur in the N protein region, and the efficiency of primers and probes against the N protein coding region will be affected by these mutations. Specific point mutations are indicated in primer or probe regions used in various countries, and these mutations may hamper the detection efficiency of RT-PCR kits and cause false-negative results. Therefore, for the detection of newly occurring mutated variants of the SARS-CoV-2 virus, updating specific primers and probes is essential for the reliability of the kits. E envelope protein, F forward primer, N nucleocapsid protein; ORF open reading frame, P probe, R reverse primer, RdRp RNA-dependent RNA polymerase.

Fig. 3

A schematic illustration of the SHERLOCK detection assay. Using nasopharyngeal swabs as an example, conventional RNA extraction is used as the input and is followed by reverse transcription and loop-mediated isothermal amplification. The CRISPR-Cas13-RNA complex is activated by binding to a complementary RNA target, while CRISPR-Cas13 exhibits nonspecific endonuclease activity, which activates and cleaves fluorescent RNA sensors. The fluorescent RNA sensor is quenched when it is intact, whereas it emits fluorescent signals when it is cleaved by the activated CRISPR-Cas13 complex.