Biocredit Antigen Test

2.1. Respiratory isolates

The SARS-CoV-2 culture isolate (strain hCoV-19/Hong Kong/VM20001097/2020, the first COVID-19 case detected in Hong Kong) was used to perform a serial tenfold dilution to determine LOD between different assays.

To evaluate the cross-reactivity of the RAD test, 13 non-SARS-CoV-2 respiratory virus isolates were tested. They were influenza A(H1pdm09), influenza A(H3), influenza B, adenovirus, coronavirus type OC43, coronavirus type 229E, parainfluenza virus type 1, parainfluenza virus type 2, parainfluenza virus type 3, parainfluenza virus type 4, respiratory syncytial virus, rhinovirus and enterovirus.

2.2. Respiratory samples

From February 1, 2020 to April 21, 2020, respiratory samples from individuals confirmed with SARS-CoV-2 infection by RT-PCR targeting the SARS-CoV-2 virus–specific RdRp gene were retrieved for this evaluation. Samples were placed in viral transport media (VTM) or Phosphate-Buffered Saline (PBS) for RNA extraction. The remaining part of the suspension was stored at −70 °C until use in this study. Virus concentrations in samples were estimated from cycle threshold (Ct) value.

The Public Health Laboratory Services Branch (PHLSB) in Hong Kong has been designated as WHO COVID-19 reference laboratory since April 2020 and all confirmed cases in Hong Kong were either diagnosed or confirmed by PHLSB [4]. Total number of 368 confirmed COVID-19 samples collected with sufficient quantity were available for this study in descending order: throat saliva (N = 122), nasopharyngeal swab and throat swab (NPS & TS, N = 103), nasopharyngeal aspirate and throat swab (NPA & TS, N = 81), sputum (N = 62).

2.3. RAD kits for SARS−CoV-2 detection

We evaluated the only commercially available RAD kit in Hong Kong at the time of starting the evaluation, BIOCREDIT COVID-19 Ag for the diagnosis of SARS-CoV-2 infection.

2.4. BIOCREDIT COVID-19 Ag

The intended use for the BIOCREDIT COVID-19 Ag kit is for nasopharyngeal swab sample. As NPA & TS, NPS & TS, sputum and throat saliva had either been eluted in VTM or suspended in PBS, the test was carried out with modified sample processing methods.

2.5. Sample processing by BIOCREDIT COVID-19 Ag

The recommended sample volume by the BIOCREDIT COVID-19 Ag kit was 90–150 μL. To unify the sample volume, 100 μL sample volume was used. We evaluated two sample processing methods based on the nature of the samples.

2.6. Viral culture for SARS-CoV-2 virus

Viral culture was conducted by inoculating samples onto Vero E6 cells. When virus-induced cytopathic effect was examined, identification of SARS-CoV-2 virus in culture fluid was confirmed by the RT-PCR.

2.7. RT-PCR for SARS-CoV-2 virus

The in-house developed RT-PCR was used to detect the presence of SARS-CoV-2 virus nucleic acid in all samples. It was conducted using NxtScript Enzyme and Master Mix (Roche Diagnostics GmbH, Germany). Each 10 μL reaction mixture contained 5 μL RNA samples, 2 μL Reaction Mix (5X), 0.06 μL Adpta Taq DNA polymerase (50U/μL), 0.05 μL NxtScript RT Enzyme (85U/μL), 0.9 μL volume of working primer/probe mix and nuclease-free water to obtain a final volume of 10 μL. The working primer/probe mix was prepared by mixing equal volume of forward primer, NCOV-F4: 5′-GTTGGACTGAGACTGACCTTAC-3′ (10 μM); reverse primer, NCOV-R4: 5′−CCCTAGGATTCTTGATGGATCTG-3′ (10 μM); and probe, NCOV-P4: 5′-FAM-ACAGGGTGATGATTATGTGTACCTTCCT-BHQ1−3’ (10 μM). The reverse transcription, amplification was performed in the LC480 System (Roche Diagnostics GmbH, Germany) according to the following program: 1 cycle of 50 °C for 10 min, 1 cycle of 95 °C for 30 s, 40 cycles of 95 °C for 10 s and 56 °C for 30 s; and holding at 4 °C.