Immunomolecular Assay Based on Selective Virion Capture by Spike Antibody and Viral Nucleic Acid Amplification for Detecting Intact SARS-CoV-2 Particles

doi:10.21203/rs.3.rs-1374603/v1

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Immunomolecular Assay Based on Selective Virion Capture by Spike Antibody and Viral Nucleic Acid Amplification for Detecting Intact SARS-CoV-2 Particles

https://doi.org/10.21203/rs.3.rs-1374603/v1

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Background: Effective therapeutics and vaccines for coronavirus disease 2019 (COVID-19) are currently lacking because of the mutation and immune escape of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Based on the propagation characteristics of SARS-CoV-2, rapid and accurate detection of complete virions from clinical samples and the environment is critical for assessing infection risk and containing further COVID-19 outbreaks. However, currently applicable methods cannot achieve large-scale clinical application because of factors such as the high viral load, cumbersome virus isolation steps, demanding environmental conditions, and long experimental periods. In this study, we developed an immunomolecular detection method combining capture of the viral spike glycoprotein with monoclonal antibodies and nucleic acid amplification via quantitative reverse transcription PCR to rapidly and accurately detect complete virions.

Results: After constructing a novel pseudovirus, screening for specific antibodies, and optimizing the detection parameters, the assay achieved a limit of detection of 9 × 10² transduction units/mL of viral titer with high confidence (~95%) and excellent stability against human serum and common virus/pseudovirus. The coefficients of variation were 1.0–2.0% for intra-assay and inter-assay analyses, respectively. Compared with reverse transcription-PCR, the immunomolecular method more accurately quantified complete virions. SARS-CoV-2/pseudovirus was more stable on plastic and paper than on aluminum and copper in the detection of SARS-CoV-2 pseudovirus under different conditions, and complete virions were detected up to 96 h after they were applied to these surfaces (except for copper), although the titer of the virions was greatly reduced.

Conclusions: Convenient, inexpensive, and accurate complete virus detection can be applied in many fields, such as monitoring the infectivity of convalescent and post-discharge patients and assessing high-risk environments (isolation rooms, operating rooms, patient living environments, and cold chain logistics). This method can also be used to detect intact virions such as those of hepatitis B and C viruses, human immunodeficiency virus, influenza, and the partial pulmonary virus, which may further improve the accuracy of diagnoses and facilitate individualized and precise treatments.

COVID-19

complete virions

Spike

infection risk

immunomolecular detection

Coronavirus disease 2019 (COVID-19), caused by the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a serious global public health threat[1, 2]. Despite the extensive financial investments in the diagnosis, treatment, and prevention of COVID-19, the pandemic is ongoing because of the high infectivity, pathogenicity, mutation and immune escape of SARS-CoV-2[3–6].

The novel β-coronavirus, SARS-CoV-2, contains a single-strand positive-sense RNA genome of 30 kilobases containing five major open reading frames (ORFs): replicase complex (ORF1ab), spike (S), envelope (E), membrane (M), and nucleocapsid (N), and is approximately 80–120 nm in diameter[7–9]. SARS-CoV-2 enters and infects the host cell via the S protein on the outer viral membrane by interacting with the host receptor human angiotensin-converting enzyme 2 (hACE2)[10–12]. The positive single-stranded RNA is released from the virus and moves into the cell nucleus, where nonstructural proteins, viral polymerases, RNA, and viral structural proteins are synthesized sequentially. These components are then packaged into new complete virion particles. The progeny virus is wrapped in a vesicle in the cytoplasm and released from the cell through exocytosis to begin the next viral cycle of infection[13]. Analogously, the nucleocapsid protein can form condensates with viral genomic RNA which are secreted extracellularly as “subviral particles and is a common occurrence in the life cycle of human viruses[14–16]. For example, the core protein and viral DNA of hepatitis B virus (HBV) can assemble as capsid particles that are secreted, and the capsid protein (CA) of human immunodeficiency virus (HIV) and core protein of hepatitis C virus can coat viral RNA and emit it as sub-virus particles[17–20]. Based on the life cycle of SARS-CoV-2, intact virus particles clearly represent the only elements that sustain infection, whereas subviral particles cannot sustain the viral life cycle consistently enough to persistently infect the host.

According to previous studies, human-to-human transmission via respiratory droplets and close contact is the main transmission route of SARS-CoV-2[21, 22]. However, some cold chain practitioners have been infected via contact with contaminated cold chain goods globally; therefore, contact with objects contaminated by SARS-CoV-2 may also cause infection[23–25]. Recent studies reported that positive results for COVID-19 were detected in blood samples of convalescent patients and different environmental conditions, including cold-chain logistics, hospital rooms, and airtight cabins[26–31]. Whether positive signals indicate that the patient or environment is at a high risk of infection, as well as the influence of subviral particles and free RNA fragments, requires further analysis.

Several methods have been developed to ensure the timely and effective detection of SARS-CoV-2 infection, including pathology-based chest computed tomography, protein-based viral antigens, and antibody detection and nucleic acid-based RNA detection[32–41]. These assays are important for diagnosing coronavirus infections and forming clinical treatment prognoses[42]. However, some “subviral particles” are either missing both the genome and capsid or missing only the genome, resulting in false-positive detection signals that do not truly reflect the viral replication and infection abilities. For example, HBV subviral particles composed of only viral surface proteins are present in the blood of infected individuals at up to a 100,000-fold excess compared to complete virions (at 10¹⁴/mL). Empty (genome-free) virions, which contain the surface proteins enclosing the viral capsid but no genome, exhibit 100-fold higher levels over complete virions in the blood of infected individuals (10¹¹/mL)^[43–46]. Therefore, detecting intact SARS-CoV-2 particles in body fluid (including blood, saliva, etc)and the environment is critical for assessing infection risk and containing further outbreaks of COVID-19.

Using pseudoviruses enables research of highly pathogenic viruses without the requirement for highly secure biosafety facilities, which has greatly benefited research on COVID-19[47–50]. However, the previously reported SARS-CoV-2-related recombinant virus either simulated the functional structure of S and N protein or incorporated fragments of its nucleic acids, and failed to simulate the functional structure of complete SARS-CoV-2 particles. Here, we constructed a SARS-CoV-2 pseudovirus system to simulate the functional structure of SARS-CoV-2 and obtained monoclonal antibodies with high affinity through screening of 11 strains of specific antibodies directed to S proteins. An immunomolecular assay was developed by combining immunocapturing SARS-CoV-2 spike protein and nucleic acid amplification via quantitative reverse transcription polymerase chain reaction (RT-qPCR) for highly sensitive and selective detection of intact SARS-CoV-2 particles.

Construction of pseudo-SARS-CoV-2 virus and screening of specific antibodies directed to Spike glycoproteins

To simulate the functional structure of intact SARS-Cov-2 particles, we constructed a SARS-CoV-2 pseudovirus expressing the Spike glycoproteins outside the envelope and incorporating four viral uncompleted genes of SARS-CoV-2 (ORF1ab, ORFab, N, and M) into the lentivirus encoding green fluorescence protein (GFP) (Figure 1a). A lentivirus-transformed plasmid, pLV-SARS-CoV-2-F1abFabNE-GFP, and an envelope plasmid, pCMV3-2019-nCoV-Spike (S1+S2), were constructed first (Figure S1). After generating SARS-CoV-2 pseudoviruses through co-transfection, we tested their infectivity by transducing HEK-293FT-hACE2 cells (Figure S2). Abundant GFP fluorescence was observed via fluorescence microscopy at 48 and 72 h, suggesting successful construction and transduction of the pseudovirus (Figure 1b). The efficiency of incorporating the SARS-CoV-2 S protein and targeted sequence into the lentiviral cells was evaluated using monoclonal mouse anti-S (S2 domain) via western blotting and qRT-PCR targeted to the F1ab gene, respectively (Figure 1c and 1d). Specific bands at 190 and 80 kDa corresponded to the monomer S protein (S1 +S2) and S2 domains, respectively (Figure 1c, lane 3), and the positive signal for the F1ab gene supported the functional integrity of the SARS-CoV-2 pseudovirus.

Based the SARS-CoV-2 pseudovirus, 11 monoclonal antibodies (CQ2, CQ20, CQ25, CQ8, CQ12, CQ001, CQ100, CQ040, CQ042, CQ023, and M1E1) directed to the Spike glycoproteins of SARS-CoV-2 were prepared and screened according to their affinity using western blotting and particle gel assay[51]. Influenced by its natural shape and charge, the active Spike glycoproteins of SARS-CoV-2 only could be detected with the CQ2, CQ25, and M1E1 antibodies (Figure 1e and 1f). Among these 11 candidates, the CQ25 antibodies showed excellent affinity and specificity for S protein.

Immunomolecular detection platform for SARS-CoV-2/pseudovirus particles

After preparing the SARS-CoV-2 pseudovirus and high-affinity antibodies, an immunomolecular detection platform for intact SARS-CoV-2/pseudovirus was designed. In summary, carboxyl groups were coated on the surface of nanomagnetic beads that were covalently bound to the amino groups of the antibodies to form peptide bonds, resulting in the generation of magnetic beads-antibodies complexes. After co-incubation at 20~25 ℃, anti-S antibodies-magnetic bead complexes specifically captured the SARS-CoV-2/pseudovirus particles. Following separation of the supernatant using a magnetic separator, the SARS-CoV-2/pseudovirus particles were enriched and separated from other “subviral particles” without S glycoprotein and free RNA fragments. Finally, nucleic acid-based detection via qRT-PCR excluded the influence of empty (genome-free) virions to further ensure that the detection platform only targeted intact SARS-CoV-2/pseudovirus particles (Figure 2a).

To establish an immunomolecular detection platform for intact SARS-CoV-2 particles, carboxyl magnetic beads and anti-S antibody (CQ25) were coupled. The effect of conjugation was evaluated by measuring the protein concentration of the complexes following conjugation using a BCA assay (Figure 2b) and particle size analysis (Figure 2c). Compared with untreated carboxyl magnetic beads, the complexes showed much higher absorption intensities (approximately 10-fold) at 562 nm and much larger average effective diameters (approximately 2-fold), indicating that the anti-S antibodies were successfully conjugated with the magnetic beads. The complexes and corresponding supernatant were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie bright blue staining, which confirmed the successful coupling and optimization of the coupling parameters (Figure 2d). Next, we verified whether the SARS-CoV-2 pseudovirus could be captured by anti-S antibody-magnetic beads complexes (MB-CQ25) at the protein and nucleic acid levels. Because HIV1 P24 is the most abundant marker protein in the lentivirus capsid, we identified the viral capsid proteins of captured virions in a viral particle gel assay with the monoclonal mouse anti-HIV 1 p24 antibody. As shown in Figure 2e, specific bands were observed in the complexes following capture. Importantly, compared with the negative control samples, including the SARS-CoV-2 S pseudovirus, VSV-G pseudovirus, and HBV, only our SARS-CoV-2 pseudovirus generated a positive signal via nucleic acid-based detection following capture (Figure 2f). These results show that the immunomolecular detection platform specifically detected intact SARS-CoV-2/pseudovirus particles.

Immunomolecular assay detects only intact virus particles

To verify the detection target of the immunomolecular assay, sucrose density gradient ultracentrifugation was performed to isolate the pseudovirus particles[46]. As shown in Figure. 3a, the S glycoprotein peaked in the 10–20% sucrose gradient according to western blot analysis, whereas the pseudovirus particles settled at the 40% sucrose gradient in the gel assay, suggesting the presence of abundant free S proteins (large excess over complete virions) in the cell supernatant. Additionally, qRT-PCR and immunomolecular assays were used to determine the RNA levels of total and intact virions in different density gradients. The data indicated that pseudovirus particles were enriched in the 40% sucrose gradient fraction. Nevertheless, the virogene level was significantly higher than that of the intact virion (P < 0.05), suggesting that some subviral particles (without S envelope proteins) were produced during virus packaging; these results are consistent with previous reports on HBV[44, 45]. Using different titers of intact virions to transduce 293FT-HEK-hACE2 cells, we found that the titer of intact virions detected in the immunomolecular assay was positively correlated with infectivity, suggesting that the detection target of the immunomolecular assay is intact virus particles (Figure 3b).

Validation of immunomolecular assay for intact SARS-CoV-2 particles

After optimizing the detection conditions, ten-fold gradient dilutions were prepared and analyzed using fluorescence qRT-PCR in parallel to determine the linear range (Figure S3). When the titer ranged between 6 × 10² and 6 × 10⁷ transduction units (TU)/mL, there was a linear relationship between the Cq value and its titer (log_10-transformed) in the immunomolecular assay (R² = 0.99) (Figure 4a). As shown in Figure 4b, there were significant differences between the negative samples with the 24 carboxy magnetic beads coupled with CQ25 antibodies (MB) and positive samples containing 24 instances of captured pseudovirus (P < 0.0001). Using a mean titer of the negative samples of +1.96 standard deviation, the limit of detection was 900TU/mL. Although the Cq value could be measured when the pseudovirus titer was lower than 900 TU/mL, the results of fluorescence quantitative PCR were negative.

Human serum contains a large amount of albumin and various antibodies, which may affect the assay stability. Additionally, if the patient carries other non-specific viruses, the specificity and stability of the method may be affected. To evaluate these factors, the HBV supernatant collected from AD38 cells and patient serum with different titers of HBV were used to verify the anti-interference ability of this immunomolecular assay. As shown in Figure 4c, for the low-titer group, adding HBV and patient serum with different titers of HBV had a negligible effect on the quantitative qualitative detection rate of intact SARS-CoV-2 pseudovirus particles, with coefficients of variation of 1.5% and 2.0% for intra-assay and inter-assay analyses, respectively; those of the high-titer group were 1.4% and 1.0%. Thus, this method is significantly stable against non-specific viruses and human serum. We further examined the stability of the SARS-CoV-2 pseudovirus over time on various surfaces, including copper, aluminum, paper, and plastic, to mimic different environmental samples (Figure 4d). SARS-CoV-2 was more stable on plastic, aluminum, and paper than on copper, and viable virus was detected up to 96 h after application to these surfaces; however, the virion titers were greatly reduced (from 10^7.38 to 10^4.05 TU/mL onaluminum, from 10^7.38 to 10^4.58 TU/mL on paper, and from 10^7.38 to 10^4.84 TU/mL on plastic). Notably, although the total RNA produced a positive detection signal, live virus was not detected on the copper surface at 72 h. Moreover, the gap in the titer between the total RNA and intact virions increased over time under different conditions. These results suggest that RNA fragments had different stabilities under different conditions after the complete virions were degraded, supporting RNA levels alone should not be used to assess infection risk.

The COVID-19 pandemic has caused an unprecedented world public health crisis. Currently, effective therapeutics for COVID-19 are lacking, and vaccine development and application cannot keep pace with the high mutation rate of SARS-CoV-2. The diagnosis of SARS-CoV-2 is critical for controlling the spread of COVID-19. Traditional detection methods based on nucleic acid fragments or protein are affected by “subviral particles,” such as free RNA fragments, free antigens, nucleocapsid protein-RNA condensates, and empty virions, and thus have failed to provide an accurate basis for patient medication and environmental assessment. Previously, a platform coupled to Raman spectroscopy was used to capture viruses based on their size; however, the nonspecific and harsh experimental conditions prevented application of this approach in clinical laboratories[52]. The median tissue culture infective dose was also used to quantify virus titers and assess infectivity. However, the high viral load, cumbersome virus isolation steps, demanding environmental conditions, and long experimental periods made it difficult to apply this method in clinical laboratories[53–55]. As free antigens in the supernatant are present in much greater excess over complete virions, detection of viral particles based on viral antigens cannot completely reflect the infection risk of patients and the environment[37, 39]. Here, we developed an immunomolecular assay for detecting intact SARS-CoV-2 particles. This method achieved excellent stability and specificity against other common human virus/pseudovirus and excellent accuracy and convenience for detecting intact SARS-CoV-2 particles in environmental samples. Compared to previously reported pseudoviruses, our designed pseudo-SARS-CoV-2 virus better simulated intact SARS-CoV-2 particles at the nucleic acid and protein level. Four fragments of viral RNA sequences, ORF1ab (15415–15540), ORFab (12750–13491), N gene (28750–29150), and E gene (26360–26381), wrapped in the genome of the pseudovirus can be detected using all nucleic acid detection reagents recommended by the World Health Organization and may be sold on the market without redesigning the primers used in qRT-PCR. In addition, compared with previously reported SARS-CoV-2 biomimetic virus-like particles with ∼16% variation, our novel pseudovirus shows much higher stability and may serve as a quantitative diagnostic tool for SARS-CoV-2 (Figure S4)[56].

This assay can be used in a variety of scenarios, such as for monitoring the infectivity of convalescent and post-discharge patients and assessing the risk of infection in high-risk environments including virus detection sampling houses, international flight cabins, and international cold-chain logistics bases. For research and applications, affinity antibodies and carboxyl magnetic beads can be optimized to improve the detection sensitivity. Moreover, isothermal nucleic acid amplification methods may be combined with this detection method to further simplify the experimental process and reduce professional dependence on experimental equipment, enabling applications of this method in clinical practice. This immunomolecular assay can also be used to detect other intact virus particles such as HBV, hepatitis C virus, HIV, influenza, and the partial pulmonary virus, which may improve the accuracy of diagnoses of these infectious diseases and facilitate individualized and precise treatments. However, the potential applications for detecting these intact virus particles require further experimental studies.

We prepared pseudovirus that fully simulates the SARS-CoV-2 virus in a safe and convenient manner. After screening monoclonal antibodies with high affinity for S protein, we developed an immunomolecular assay combined immunocaptured of SARS-CoV-2 S protein and nucleic acid amplification via qRT-PCR for highly sensitive and selective detection of intact SARS-CoV-2 particles. This method enables accurate assessment of the contagiousness of infected patients and/or potential infection hazard in high-risk environments.

Production and titration of pseudoviruses

A plasmid containing partial sequences of the SARS-CoV-2 ORF1ab gene, N gene, E gene, and GFP reporter was constructed and named as PLV-SARS-CoV-2-F1abMEN-GFP. Pseudoviruses were produced and titrated using methods similar to the Rift Valley Fever pseudovirus, as previously described^[57]. Briefly, HEK-293FT cells were transfected with the plasmids pCMV3-2019-nCoV-Spike(S1+S2), pLV-SARS-CoV-2-F1abFabME-GFP, and pMD2.G (Sino Biological, Inc. Beijing, China) using Lipofectamine 8000 (C0533, Beyotime Biotechnology, Shanghai, China). The virus supernatant was collected and mixed at 48 and 72 h after transfection and centrifuged at 4 °C at 4000 ×g for 10 min to remove cell debris. The culture supernatant was placed on a 20% sucrose solution and centrifuged at 25,000 rpm (112,000 ×g) for 15 h at 4 °C in a Beckman SW28 rotor (Beckman Coulter, Brea, CA, USA). For this HIV 1 pseudovirus system, the titer of PsVs was quantified using an HIV-1 Gag p24 DuoSet ELISA kit (KIT11695, Sino Biological Inc. Beijing, China) according to the manufacturer’s instructions.

After transfection with lentivirus overexpressing hACE2 or an empty vector at 48 h, 1 × 10⁵ HEK-293FT cells in a 96-well plate were transduced with the SARS-CoV-2 pseudovirus and control pseudovirus encoding GFP at a multiplicity of infection of 2 vp/cell for 24 h at 37 °C. Infection was visualized using a fluorescence microscope at 48 and 72 h.

Conjugation of carboxyl magnetic beads with antibody

After continuous activation with N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide solution at 25 °C for 30 min, carboxymethyl magnetic beads (MSP-COOH-F1 18030106, FlyW&Y Bio, Chongqing, China) were added to the diluted CQ25 antibodies, mixed, and gently rotated at 4 °C for 4 h. The magnetic bead–antibody complexes and supernatant were separated using a magnetic separator. The complexes were blocked gently with a 1% bovine serum albumin solution at 25 °C for 30 min. SDS-PAGE and Coomassie blue staining were performed to evaluate the coupling effect using coupled magnetic beads and separate the supernatants.

qRT-PCR

SARS-CoV-2 RNA levels were detected using qRT-PCR on a CFX96 system (Bio-Rad, Hercules, CA, USA) using a 2019-nCoV nucleic acid detection kit (fluorometric real-time PCR) (001, Sansure Bio, Hunan, China). Each sample was evaluated in triplicate, and two no-template control wells were included to confirm that there was no contamination.

Western blotting

The samples were mixed with 6× SDS sample buffer, boiled for 10 min at 95 °C, and subjected to SDS-PAGE and immunoblotting. The pseudovirus was detected using mouse/human anti-S/membrane antibodies (CQ2, CQ20, CQ25, CQ8, CQ12, CQ001, CQ100, CQ040, CQ042, CQ023, and M1E1, a gift from the Laboratory of Molecular Biology of Infectious Diseases, Chongqing Medical University, Chongqing, China) at a 1:1000 dilution. Goat anti-mouse IgG (No. SA00003-1, Proteintech, Rosemont, IL, USA) was used at a 1:4000 dilution as the secondary antibody.

Viral particle gel

Denatured samples were resolved on a 1% agarose gel in 1× TAE buffer at 70 V for 2 h. Particles in the gel were transferred to a nylon membrane in 1× TBE buffer for 12 h based on the Siphon principle[51]. After transfer, the membrane was blocked in 5% bovine serum albumin for 30 min and incubated in an anti-HIV-1 P24 antibody solution or anti-S antibody solution for 12 h at 4 °C. The membranes were exposed following washing with 1× Tris-buffered saline containing 1‰ Tween 20.

Particle size analysis

The effective particle size of the carboxyl magnetic beads (MB) and magnetic beads-CQ25 antibody complex (MB-CQ25) were characterized using a NanoBrook 90PLUS PALS particle size analyzer (Brookhaven Instruments Corporation, Holtsville, NY, USA). The sample was added to pure water (1‰ Triton-100) and mixed to ensure even dispersion in the medium. If necessary, the sample was sonicated for 15 min. The particle size was determined after setting the parameters according to the instrument manual. Three complex holes were placed each time, and this process was repeated twice. The data were analyzed and plotted using GraphPad Prism 8 software (GraphPad Inc., San Diego, CA, USA).

Sucrose density gradient ultracentrifugation

Sucrose density gradient centrifugation was performed as previously described[46]. Briefly, discontinuous sucrose density gradients (10%, 20%, 30%, 40%, 50%, and 60%) were prepared in PBS. The culture supernatant was laid on the linear sucrose gradient and centrifuged at 25,000 rpm (112,000 ×g) for 15 h at 4 °C in a SW28 rotor (Beckman Coulter).

Statistical analyses

Linear regression, descriptive statistics, repeated measures analysis of variance, and two groups of unpaired t-tests were performed using the statistical software package SPSS version 21.0 for Windows (SPSS Inc., Chicago, IL, USA). All tests of significance were two-tailed, and statistical significance was set at P < 0.05.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

All data generated or analyzed during this study are included in the article.

Competing interests

The authors declare that they have no competing interests.

Funding

This work was supported by grants from the National Science and Technology Major Project (2017ZX10202203 and 2018ZX10732202) (to AH), the Chongqing Natural Science Foundation (CSTC2018jscx-msybX0032 and CSTC2016jcyjA0204) (to DW), and the Natural Science Foundation of Chongqing Yuzhong (20190145) (to DW).

Authors’ contributions

X. L, J. L, and H. Z contributed equally to this work.

Acknowledgements

We thank Prof. Aishun Jin (Department of Immunology, Chongqing Medical University, China) for providing some antibodies. We would like to thank Editage (www.editage.cn) for its linguistic assistance during the preparation of this manuscript.

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No competing interests reported.

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Editorial decision: Major revision
13 May, 2022
Reviews received at journal
10 Mar, 2022
Reviewers agreed at journal
18 Feb, 2022
Reviewers invited by journal
18 Feb, 2022
Editor assigned by journal
10 Feb, 2022
Submission checks completed at journal
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Immunomolecular Assay Based on Selective Virion Capture by Spike Antibody and Viral Nucleic Acid Amplification for Detecting Intact SARS-CoV-2 Particles

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