Primer and probe design for RT-qPCR.
To detect multiple CoVs including SARS-CoV-2 using a one-step multiplex RT-qPCR, primers and probes were designed based on the 265 reference of published coronavirus sequences (isolated from 2005 to 2020) in GenBank and Global Initiative on Sharing All Influenza Data (GISAID) database. The sequences were aligned using ClustalW in BioEdit software (version 6.0) and used as a template for the design of the multiplex RT-qPCR. The probes were labelled with FAM (5-Carboxyfluorescein) and HEX (5-hexachloro-fluorescein) for detection of the SARS-CoV-2 and pan-SARS-like CoVs on M gene, respectively. And Cy5 (Cyanine Dye 5) and Texas Red for detection of the SARS-CoV-2 and Pan-SARS-CoVs on S2 domain of S gene, respectively. All primers and probes were synthesized by Cosmogenetech Inc. (Seoul, Republic of Korea) and detailed information regarding all the designed primers and probes including WHO guidelines [2] are presented in Table 1.
Viruses and viral RNA samples
To evaluate the sensitivity and specificity of our multiplex RT-qPCR, cell culture supernatants from cells infected with coronaviruses and other respiratory viruses were used. In total eighteen different cultured viruses were used. Korean isolated four of SARS-CoV-2 (wild type, alpha variant, beta variant, and delta variant), human coronavirus-NL63 (Korea/CN0601/14), human coronavirus-229E (Korea/KUMC-9), and MERS-CoV (MERS-CoV/KOR/KNIH/002_05_2015 were obtained from the Korea Centers for Disease Control and Prevention (KCDC, Republic of Korea), and other viruses were obtained from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) or purchased from the Korea Bank for Pathogenic Viruses (KBPV, Republic of Korea) [16]. The RNA of SARS-CoV (HKU-39849) was provided by Dr. Seungtaek Kim[22] and viral RNA samples of bat-derived SARS-related CoV (GenBank accession number: MK991935, MK991936) from bat fecal samples that were used in the specificity assays described in table 2 were provided by Prof. Hye Kwon Kim[21].
SARS-CoV-2 virus propagation
VERO cells were purchased from the American Type Culture Collection (CRL-1586) and were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Corning, NY, USA) containing 5% Fetal Bovine Serum (FBS, Thermo Fisher Scientific, MA, USA) and Penicillin (100 IU/ml, Thermo Fisher Scientific), and incubated at 37 °C in 5% CO2. For the titration of the SARS-CoV-2 virus, VERO cells were seeded at 1 × 105 cells per well in 96-well-plates for 24 h before inoculation with serial, ten-fold dilutions of SARS-CoV-2. After 72 h incubation, TCID50/ml was determined using the method described by Reed and Muench [24]. All SARS-CoV-2 experiments were conducted at KRIBB (Daejeon, Republic of Korea) under the approval and in accordance with the guidelines of the Institutional Biosafety Committee (IBC, approval number KRIBB-IBC-20200208) of KRIBB. Experimental work with SARS-CoV-2 was conducted in a biosafety level-3 (BL-3) facility at KRIBB (permission number KCDC-HP-19-3-01).
One-step multiplex RT-qPCR analysis
Viral RNA was purified using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions, and samples were stored at -80°C until further analysis. Viral RNA of SARS-CoV-2 was extracted in a BL-3 containment facility in KRIBB and all RT-qPCR were conducted in BL-2 facilities at the KRIBB. The RT-qPCR assays were performed using the LightCycler 96 instrument and analysis software (Roche, Switzerland). The one-step RT-qPCR reaction mixture included 5 μl of RNA template, 10 μl of 2X reaction buffer supplemented with the SensiFAST Probe No-ROX One-Step Mix (Bioline, UK), 0.2 μl of reverse transcriptase (Bioline, UK), 0.4 μl of RNase inhibitor (Bioline), 1 μl of each primer (10 pmol), 1 μl of each probe (5 μM), and RNase-free water to add up to a final volume of 20 μl. PCR cycling conditions used were as follows; initial at 45°C for 15 mins and 95°C for 10 mins for the reverse transcription step, followed by 40 cycles at 95°C for 5 s and 58°C for 30 s.
Determination of sensitivity and specificity of the RT-qPCR analysis
To assess the sensitivity of the one-step multiplex RT-qPCR assay, SARS-CoV-2 was serially diluted 10-fold from 100 to 106 TCID50/ml and then viral RNA was extracted from these samples using the QIAamp Viral RNA Mini Kit according to the manufacturer’s instructions (Qiagen). The intact viral RNA was then amplified by RT-qPCR. After RT-qPCR, the presence of amplicons was confirmed using 2% agarose gel electrophoresis and Sanger sequencing. To optimize the specificity of one-step multiplex RT-qPCR for SARS-CoV-2 detection, different intact viral RNA templates were used from human coronaviruses (SARS-CoV, wildtype of SARS-CoV-2, three of SARS-CoV-2 variants, MERS-CoV, hCoV-229E and hCoV-NL63), two bat-derived SARS-related coronaviruses, influenza A viruses (A/California/04/09, A/Korea/37/2012) and B virus (B/Brisbane/60/2008); and five other swine/human pathogens, including Porcine epidemic diarrhea virus (PEDV/Korea/SM98), Dengue type 2 (Korea/DENV-2/KBPV-VR-29); Parainfluenza virus type 1 (Korea/KUMC-44); Human Respiratory Syncytial virus (HRSV-A/IC688/12); and Adenovirus type 3 (KUMC-62).
Collection of human clinical samples
Virus-negative nasal swabs and sputum samples were acquired from healthy donors and clinical samples were obtained from COVID-19 patients in the Chungnam National University School of Medicine (Daejeon, South Korea). A total of 67 clinical samples (40 males and 27 females) were used in this study, including 29 cases of laboratory-confirmed COVID-19 that tested positive for SARS-CoV-2 RNA by the RdRp2 assay and 38 cases with healthy donors (ranging in age from 18 to 64 years old). Written informed consent was obtained from all human subjects who participated in this investigation. All samples were collected under the approval of the Institutional Review Board of the National Medical Center, Republic of Korea (IRB number: 2020-03-057). All experiments using human clinical samples were performed following approved guidelines.