This study was approved by the Institutional Review Board of the Faculty of Medicine Siriraj Hospital, Mahidol University (SIRB protocol 463/2563(IRB4); COA: Si 503/2020).
Respiratory samples, mainly nasopharyngeal and throat swabs, were collected from 454 suspected COVID-19 cases, including pre-operative patients at Siriraj Hospital, Mahidol University, Bangkok, Thailand, from March to May 2020. Samples were mixed in 2 mL of viral transport media (VTM), consisting of Hanks’ balanced salt, 0.4% fetal bovine serum, HEPES, antibiotic and antifungal agents. Samples were transported at 2-8 °C to the Microbiology laboratory, Siriraj Hospital, for processing within a few hours. All specimens were processed in biosafety level-3 (BSL-3) and biosafety level-2 enhanced (BSL-2 +) facilities with full personal protective equipment.
See more: Covid 19 rdrp gene range
Viral RNA Extraction
MagLEAD 12gC automated extraction platform (Precision System Science, Chiba, Japan) was used to extract SARS-CoV-2 RNAs from 200 µL of nasopharyngeal and throat swabs. Extraction was performed according to the manufacturer’s instructions. Viral RNA was eluted with 100 µL buffer and used for RT-PCR assay.
SARS-CoV-2 RNA detection using real-time RT-PCR
Allplex™ 2019-nCoV Assay (Seegene, Korea), which targets envelope gene (E) of Sarbecovirus, and RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes of SARS-CoV-2, was used for SARS-CoV-2 RNA detection according to the manufacturer’s instructions. Briefly, 8 μL of extracted RNA was added to 5 μL of 5X Real-time One-step Buffer, 5 μL of 2019-nCoV MuDT Oligo Mix (2019-nCoV-MOM), 2 μL of Real-time One-step Enzyme, and 5 μL of RNase free water. The CFX-96 real-time thermal cycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used for amplification. The conditions consisted of 1 cycle of 20 min at 50 °C, 1 min at 95 °C and followed by 45 cycles of 15 s at 94 °C, 30 s at 58 °C. The result was analysed using Seegene Viewer (Seegene, Korea), in which a cycle threshold value (Ct-value) < 40 for all three target genes was defined as a positive result.
Rapid SARS-CoV-2 antigen detection assay
Standard Q COVID-19 Ag test (SD Biosensor®, Chuncheongbuk-do, Republic of Korea) is a rapid chromatographic immunoassay for the detection of SARS-CoV-2 nucleocapsid (N) antigen in respiratory specimens. This rapid antigen test device has two pre-coated lines on the result window: control (C) and test (T) lines. The control (C) region is coated with mouse monoclonal anti-chicken Igγ antibody; the test (T) region is coated with mouse monoclonal anti-SARS-CoV-2 antibody against SARS-CoV-2 N antigen. Detectors for SARS-CoV-2 N antigen presented in the specimen are mouse monoclonal anti-SARS-CoV-2 antibody conjugated with color particles. The antigen-antibody color particle complex migrates via capillary force and is captured by the mouse monoclonal anti-SARS-CoV-2 antibody coated on the test (T) region. The colored test (T) line’s intensity depends on the amount of SARS-CoV-2 N antigen presented in the sample.
This rapid Ag test kit was used for the detection of SARS-CoV-2 antigen in respiratory samples in this study. Specimens were processed in biosafety level-3 (BSL-3) and biosafety level-2 enhanced (BSL-2 +) facilities. Five to ten glass beads were added to the samples in VTM tubes. For highly viscous samples, additional VTM was added to reduce the viscosity. Specimens were mixed using a vortex mixer to disrupt thick mucus. The 200 μL of each nasopharyngeal and throat swab specimen was added to the extraction buffer provided in the kit. The filter nozzle cap was pressed tightly onto the extraction tube. Three drops of the extracted sample were applied on a test device, and the test result was read in 15-30 min. For positive COVID-19 antigen result, two colored lines of control (C) and test (T) lines were presented.
Descriptive statistics were used to describe general information of patients. Continuous data were presented in mean, standard deviation (SD), median, and range. Categorical data were presented in numbers, percentages, and 95% confidence interval (95% CI). Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) were calculated using an online statistical tool .