Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Antigen Detection Kit
Cases of coronavirus disease 2019 (COVID-19) in Indonesia continue to rise and even more so in recent weeks. Contact tracing and surveillance are important to locate cases in the community, including asymptomatic people. The diagnosis of COVID-19 depends on the detection of viral RNA, viral antigen or, indirectly, viral antibodies. Molecular diagnosis, using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), is the common standard method; however, it is not widely available in Indonesia and requires a high-level laboratory.
Rapid point-of-care antibody tests have been widely used as an alternative; however, interpretation of the results is not simple and is now no longer used by the Indonesian government as a screening test for people travelling between locations. Therefore, the Indonesian government uses the Rapid Antigen Detection Test (Ag-RDT) as a screening test for travellers.
As a result, many people buy the kit online and perform Ag self-PDR at home. This raises the question of how safe and accurate it is to perform Ag auto-PDR at home. Before applying a test, it is suggested to investigate its sensitivity and specificity, compared to the gold standard, and its limitations. In this article, the laboratory diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is discussed, with emphasis on Ag-RDT and the recommendation of its adequate use in daily practice.
Rapid identification and effective isolation are crucial to curb the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To meet this requirement, rapid antigen detection diagnostic tests (Ag-RDT) are essential.
Between February 2020 and August 2020 we conducted a cohort study of patients with confirmed COVID-19. The clinical performance of Ag and Ag Gold rapid fluorescence immunoassay (FIA) was evaluated and compared in parallel with genomic and subgenomic real-time reverse transcription-polymerase chain reaction (RRT-PCR)-based assays. Cell cultures.
- Viral RNA Extraction
A fully automated instrument (Bio-seam, South Korea) was used to extract viral RNA using a Real-prep viral DNA/RNA kit (BioSewoom, South Korea). The extraction was performed with 200 μL of all samples according to the manufacturer’s protocol to obtain a final elution of 100 μL. Thereafter, samples were stored at -80 °C until later use for RT-PCR analysis.
- Detection of Severe Acute Respiratory Syndrome Coronavirus 2 RNA by One-Step Quantitative Reverse Transcription Polymerase Chain Reaction
A one-step RT-qPCR assay was performed to target the nucleocapsid (N) gene to detect SARS-CoV-2. The primers and probe were designed in-house. Briefly, 5 μL template was added to 4 μL 5X RT-qPCR mix (Roche), 0.5 μL 200X RT enzyme solution (Roche), 1 μL (10 pmol/μL) forward primer (nCov-NP- 572F 5′-GCAACAGTTCAAGAAATTC-3′), 1 μL (10 pmol/μL) reverse primer (nCov-NP-687R-5′-CTGGTTCAATCTGTCAAG- 3′), 1 μL (5 pmol/μL) probe (nCov- NP-661P-5′-FAM-AAGCAAGAGCAGCATCACCG-BH Q1-3′), and 7.9 μL of RNase-free water to obtain a total reaction mixture of 20 μL.
Analysis was performed on an Exicycler™ 96 (Ver. 4) real-time quantitative heating block (Bioneer, South Korea) under the following cycling conditions: 1 cycle at 50 °C for 10 min and 95 °C for 30 s followed by 45 cycles at 95°C for 5 s and 57°C for 30 s. SARS-CoV-2 sgRNAs were identified by RT-PCR as previously described.
The threshold value of the cycle (Ct value) was analyzed with the Bioneer Package software, and the sample was considered positive if a visible amplification plot was observed at Ct ≤ 35 and negative with Ct > 35. We selected the N gene to determine the load viral. For this purpose, the Ct values were converted to Log10 RNA copies/mL using the calibration curves as previously described. Results for other target genes, including gene E and RdRp along with gene N, are depicted in Supplementary.
- Detection of Severe Acute Respiratory Syndrome Coronavirus 2 by Rapid Diagnostic Tests for Antigen Detection
Samples were tested with two lateral flow assays: PCL COVID-19 Ag Rapid FIA (fluorescence immunoassay) and PCL COVID-19 Ag Gold (PCL, Inc. South Korea); both are diagnostic medical devices that use a double-antibody sandwich reaction with an immunochromatographic assay to quantitatively detect SARS-CoV-2 N-antigen in human respiratory samples. According to the manufacturer’s instructions, Ag Rapid FIA was intended to identify SARS-CoV-2 antigen in human nasopharyngeal swabs, while Ag Gold detects SARS-CoV-2 antigen in human saliva or nasopharyngeal swabs.
However, we used oropharyngeal, nasopharyngeal, and saliva samples for both assays, to allow parallel testing and comparison using different platforms. The manufacturer’s recommended instructions for use include incorporating the sample into the extraction buffer; however, we analyzed the samples in VTM, as it allowed rapid evaluation of numerous previously characterized clinical RRT-PCR samples. Based on this approach, the manufacturer was instructed to apply 100 µL of the sample immediately to the test card.
Before testing, the samples were thawed and kept at room temperature. Samples were then vortexed and transferred to the test card well with an average incubation time of 15 min at room temperature. For Ag Rapid FIA, results were observed on the PCLOK EZ automated analyzer in rapid test mode. Thereafter, the Ag Gold results were visually read and recognized by two different people, who mutually decided the result. The entire trial was performed in a biosafety level 2 facility with full personal protective equipment.
- Severe Acute Respiratory Syndrome Coronavirus 2 Cell culture and detection of infectious viruses
The 63 RRT-PCR characterized respiratory samples of SARS-CoV-2 incubated in Vero E6 cells (Korean Cell Line Bank, KCLB no. 21587), using 24-well cell culture plates with glass coverslips. Infected cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, Thermo Fisher Scientific, USA) supplemented with 2% fetal bovine serum and 1 × penicillin-streptomycin solution (Gibco, Thermo Fisher Scientific Inc., USA). Unidos) and then cultured at 37°C in the presence of 5% CO2 for 3-5 days observing daily the cytopathic effect (CPE).
Results were characterized as negative if no CPE was observed within 5 days. Additionally, viral RNA was extracted using culture supernatant and analyzed by two-pass RRT-PCR to validate SARS-CoV-2 proliferation. The entire trial was conducted at biosafety level 3 at the Gwangju City Environment and Health Research Institute.
In total, 150 samples were analyzed. Of these, 63 serial samples were obtained from 11 SARS-CoV-2 patients and 87 from negative controls. Serial respiratory samples were obtained 2 days before symptom onset (-2) through 25 days after symptom onset. Overall, for RRT-PCR positive samples (n = 51), the detection sensitivity of Ag rapid FIA and Ag Gold was 74.5% and 53.49%, respectively, with a specificity of 100%; however, for samples with low cycle threshold (Ct) values, Ag rapid FIA and Ag Gold showed a sensitivity of 82.61% (Ct ≤ 30, 5.6 log10 RNA copies/mL) and 80% (Ct ≤ 25, 6.9 log10 RNA copies/mL), respectively.
Despite low analytical sensitivity, both Ag-RDTs detected 100% infection in cell culture-positive samples (n = 15) and were highly effective at distinguishing viable samples from those with subgenomic RNA (66.66%). For both Ag-RDTs, all samples giving discordant results (RRT-PCR +ve/Ag-RDT -ve) were also negative by culture.
The data suggest that Ag-RDTs reliably detect viable SARS-CoV-2; therefore, they can serve as an important tool for the rapid detection of potentially infectious individuals.
COVID-19; SARS-CoV-2; antigen test.