The utility of SARS‐CoV‐2 nucleocapsid protein in laboratory diagnosis

Abstract Background The Coronavirus Disease 2019 (COVID‐19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), which has now become a global pandemic owing to its high transmissibility. The SARS‐CoV‐2 nucleocapsid protein tests are playing an important role in screening and diagnosing patients with COVID‐19, and studies about the utility of SARS‐CoV‐2 nucleocapsid protein tests are increasing now. Methods In this review, all the relevant original studies were assessed by searching in electronic databases including Scopus, Pubmed, Embase, and Web of Science. “SARS‐CoV‐2”, “COVID‐19”, “nucleocapsid protein”, and “antigen detection” were used as keywords. Results In this review, we summarized the utility of SARS‐CoV‐2 nucleocapsid protein in laboratory diagnosis. Among the representative researches, this review analyzed, the sensitivity of SARS‐CoV‐2 nucleocapsid protein detection varies from 13% to 87.9%, while the specificity could almost reach 100% in most studies. As a matter of fact, the sensitivity is around 50% and could be higher or lower due to the influential factors. Conclusion It is well suggested that SARS‐CoV‐2 nucleocapsid protein is a convenient method with a short turnaround time of about half an hour, and the presence of N antigen is positively related to viral transmissibility, indicating that SARS‐CoV‐2 N protein immunoassays contribute to finding out those infected people rapidly and segregating them from the uninfected people.

With the incessant transmission of COVID-19, SARS-CoV-2 has mutated into a variety of variants due to numerous duplications. As a result, some of these variants are highly contagious. How to prevent SARS-CoV-2 from mutating and transmitting seems to be a big problem. It is universally acknowledged that there are three basic segments in preventing infectious disease from spreading, which are eliminating the infection sources, cutting off the infection routes, and protecting the susceptible people. Truths are that utilizing a method with accuracy and rapidity could find out those who are infected with COVID-19 in a short period of time. Therefore, we could separate the infected person in time and successfully prevent the spread of this infectious disease. In a word, it is of great significance to work out the best way to rapidly screen and diagnose patient with COVID-19.
Real-time reverse transcriptase polymerase chain reaction (RT-PCR), antigen detection, and antibody detection are three different assays used to diagnose COVID-19, and they are, respectively, based on viral nucleic acid detection, viral protein detection, and human antibody detection. 4 The viral nucleic acid detection by RT-PCR is considered as the most reliable and widely used technique. 4,5 However, the nucleic acid test by RT-PCR is not convenient because it requires a molecular diagnostic laboratory equipped with trained staff and expensive equipment. Besides, another shortcoming of RT-PCR assay is the long turnaround time which limits the testing scale. 5 Viral protein detection could be detected up to 1 day ahead of clinical symptoms onset and is easy-to-use, inexpensive, and could be applied on a large scale, while the limitation is the low analytical sensitivity. 6 The benefit of antibody detection is that the device sometimes can be used at home, but positive results could prove the existence of past or current infection, or the person is vaccinated, and cross-reactivity is unavoidable. 4,7,8

| Genome structure of SARS-CoV-2
On January 10, 2020, the first whole genomic sequences of SARS-CoV-2 was published on the Virological website. Genomic analysis shows that SARS-CoV-2 is comprised of a positive-sense, singlestranded RNA genome of around 30 kb and shares 79% genome sequence identity with SARS-CoV and 87.9% with bat CoV strain bat-SL-CoVZC45 and bat-SL-CoVZXC. 2 The 5′-terminus of the genome contains ORF1a and ORF1b that encode 16 non-structural proteins (nsps1-16). 9 Most of these SARS-CoV-2 non-structural proteins have more than 85% amino acid sequence identity with SARS-CoV. 10 The 3′-terminus of the genome encodes 4 structural proteins including spike (S), envelope (E), membrane (M), and nucleocapsid (N). In addition, ORFs encode eight accessory proteins and are interspersed among these structural genes. 11 As the virus spreads, they constantly mutate their genetic code to evolve or adapt due to host immunity. 12 Most mutations in the SARS-CoV-2 genome do not affect the function of the virus, but a few mutations of SARS-CoV-2 may make the virus easier to spread, affecting how well vaccines could protect people, causing the virus less responsive to treatments for COVID-19, and/or even leading to the avoidance of SARS-CoV-2 detection, [13][14][15] which makes it difficult to implement the policy of "early diagnosis and early treatment". As the duration of the outbreak increases, mutations have occurred more frequently, potentially affecting the infectivity and pathogenicity of the virus. Table 1 compiled the commonest mutation types of SARS-CoV-2 in genomics, which demonstrates that most of these mutated sites are on structural gene S. However, different from structural gene S, the N gene is less frequently mutated, indicating that the N protein is relatively conserved and have the potential to be an interesting protein for laboratory diagnosis.

| Structural and functional analysis of SARS-CoV-2 N protein
SARS-CoV-2 N protein contains 419 amino acids, and is originated from a 1260 nucleotide length N gene after transcription and translation. 16 Sequence alignment of N protein indicates that the SARS-CoV-2 N protein closely resembles the SARS-CoV N protein rather than other human coronavirus N proteins. SARS-CoV-2 N protein consists of two structural domains named as N-terminal domain (NTD) and C-terminal domain (CTD), which are separated by a disordered linker and flanked on both termini by disordered tails. 17 The NTD, primarily responsible for RNA-binding, can be divided into three regions: a protruded basic finger, a basic palm, and an acidic wrist. 9 The CTD may function as a bridge in the formation of N protein dimer because it has been proved that CTD-CTD interaction could be found in the solution. It has been suggested that these two domains are required to bind to viral genome RNA, and then contribute to packing it into ~100 nm particles. 18,19 The disordered linker between NTD and CTD domains has a S-rich (SR) region, and could be phosphorylated at multiple sites in vitro by SRPK1, so that the N protein will be recruited to stress granules. 17 Apart from the functions mentioned above, other functions of N protein include binding with non-specific dsDNA probably by electrostatic interaction, entering the host cell, and forming the ribonucleoprotein core. 18

| UTILIT Y OF SAR S -CoV-N PROTEIN IN L ABOR ATORY D IAG NOS IS
Until late March 2022, about 48 N antigen diagnostic test kits for SARS-CoV-2 have been developed and acquired emergency use authorization from US food and drug administration (FDA). 20 Table 2 listed all authorized kits for emergency use from US FDA. 20 The most commonly used method in emergency use authorization kits for testing SARS-CoV-2 N antigen is lateral flow colloidal gold immunochromatographic assay (LF-CGIA), following by lateral flow immunofluorescence assay (LF-IFA) and chemiluminescence immunoassay (CLIA).  Many research teams have evaluated LF-CGIA for the SARS-CoV-2 N antigen, which showed that the sensitivity of LF-CGIA differs from nearly 13% to 62%, like Daniela Basso has claimed the sensitivity could be 13% while Zehra Kipritci has reported the sensitivity could reach 61.8%, yet the specificity remained rather high, almost near 100% in most experiments 22,23 (Table 3). Nonetheless, the sensitivity was around 50% in most groups, such as in a realworld comparison study in Florida, a total of 18,457 individuals were tested for the SARS-CoV-2 N antigen via LF-CGIA and RT-PCR assays for the SARS-CoV-2 RNA simultaneously. The positive percent agreement for the LF-CGIA using the RT-PCR comparator was only 49.2%. Even in symptomatic individuals, the positive percent agreement was just 51.9%. 24 In another pairing study, 3419 specimens were included to test the SARS-CoV-2 N antigen. Compared with RT-PCR assay, the LF-CGIA had a sensitivity of 64.2% for specimens from symptomatic persons and 35.8% for those from asymptomatic persons, with almost 100% specificity in specimens from both groups. 25 A study about LF-CGIA sensitivity stratified by PCR-positive cycle threshold (Ct) Ct value and time since symptom onset showed that the overall sensitivity was 78.9%, whereas for specimen obtained within 7 days after symptom onset and for specimen with a Ct value of <30, the sensitivity was 89.4% and 93.0%, respectively. 26 Another study showed that LF-CGIA for the SARS-CoV-2 N anti-

| Lateral flow immunofluorescence assay (LF-IFA)
The principle of LF-IFA is similar to that of LF-CGIA except the anti-N protein antibodies labeled with fluorescein rather than colloidal gold. 28 Accordingly, test results are identified using fluorescence intensity analyzer device rather than naked eyes. Improved LF-IFA methods, such as microfluidic immunofluorescence assay

TA B L E 2 (Continued)
test performance was improved (sensitivity = 80.0%; specificity = 98.9%). 29 In another study, Ilaria Baccani and colleagues eval- ies/ml), the MF-IFA was found to have a sensitivity of 100%. 33 It was also mentioned that sensitivity was elevated in individuals with a viral load of over log10 7 copies/ml. 32

| Enzyme-linked immunoabsorbent assay (ELISA)
ELISA is a qualitative or semi-quantitative method for the determi-   The optimal cut-off value for sample positivity was found to be 82 TCID 50 /ml, which resulting in 78% sensitivity, 73% specificity, and 77% diagnostic accuracy, whilst the optimal cut-off value for high infective risk was 106 TCID 50 /ml, and the sensitivity, specificity, and diagnostic accuracy were 94%, 96%, and 95%, respectively. 36

| Bulk Acoustic Wave (BAW) biosensor-based immunoassay
An automated Bulk Acoustic Wave (BAW) biosensor-based product from Qorvo Biotechnologies is an integrated system of instrument and reagent cartridges using immunoassay principles for the qualitative detection of the N antigens from SARS-CoV-2 in direct anterior nasal swab (NS) specimens. 39   the test results as soon as possible to avoid congestion. Thus, these places could utilize LF-CGIA and LF-IFA, and further diagnosis could be implemented during the quarantine through molecular assay in pursuit of precise results. As for some of the authorized self-test kits for SARS-CoV-2 N antigen detection mentioned above, it is convenient for the suspected people to utilize lateral flow assays to test themselves in their homes during the quarantine. There is no need for doctors to visit each home to take specimens, which avoids the possibility of transmission, and the test results could be seen within 10-20 min and could be easily interpreted by themselves. Second, in the hospital, there is a laboratory for specific instruments to have a high throughput and automatic determination of the N protein or nucleic acid of SARS-CoV-2. Furthermore, hospitals would have a place to segregate the suspected people. Therefore, it is appropriate to have those time-consuming but more precise assays including CLIA and RT-PCR in the hospital.
Moreover, through hierarchical analysis, it was found that in the earlier stage of the disease, the higher viral load the sample contains, the easier the virus could be cultured successfully, ending up in a higher sensitivity and specificity of the N antigen detection. 6,31,38 The analysis demonstrates that the transmissibility is positively related to the presence of N antigen detection. A patient with positive N antigen may be highly contagious, therefore, SARS-CoV-2 N antigen tests contribute to seeking out those infected people rapidly and dividing them from the uninfected people timely. They can be beneficial in congregate settings, such as workplace, school, or prison. 6 These places could take antigen detection into consideration. If many workers, students, or prisoners share the same symptoms and one of them has already been diagnosed with COVID-19, it is urgent for all the worker, students, or prisoners to take the SARS-CoV-2 N protein detection and they should be separated immediately, which could rapidly pick out those infected people and segregate those lucky dogs who may not be infected with COVID-19. 41 On the contrary, during the recovery period of COVID-19, antigen detection may be reliable in predicting the clearance of virus due to the sensitivity correlation with viral load, which could be applied to shortening the period of recovery isolation stage. However, this opinion still needs further clinical trials because it is only proved by a novel SARS-CoV-2 human challenge model. 42

| CON CLUS ION
In a nutshell, compared to the RT-PCR and antibody detection, antigen detection has its unique advantages and we should make full use of it. Compared to RT-PCR, it has shorter turnaround time and is free of instruments and experienced stuff, while compared to the antibody detection, it would not be influenced by the past infection and vaccination. It truly exists some drawbacks, but with time going by, its development and modification will benefit its utilization and broaden its usage, which contributes to finding out those infected people with rapidity and segregating them from the uninfected people.

AUTH O R CO NTR I B UTI O N S
XL and MX performed the data analyses and wrote the manuscript.
YL and XG contributed to the conception of the study. QD and YL contributed to the manuscript revision. All authors read and approved the submitted version.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study are available within the article.