Taqpath master mix

RNA extraction for all samples was preformed using the QIAamp 96 virus QIAcube HT kit automated platform. Our RT-qPCR reactions were carried out in a 10 μL reaction using 4 × TaqPath master mix (Thermo Fisher Scientific, Massachusetts, USA), 0.25 μM each of 2019-nCoV_N1(CDC) qPCR probe (5′-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3′), forward primer (5′-GACCCCAAAATCAGCGAAAT-3′), and reverse primer (5′-TCTGGTTACTGCCAGTTGAATCTG-3′), 4.25μL of molecular-grade H₂O, and 2.5μL of template RNA. RT-qPCR was performed on a CFX Opus 96 instrument (Bio-Rad Laboratories, Hercules, California, USA) with the following conditions: UNG incubation at 25 °C for 2 min; reverse transcription step at 50 °C for 15 min, followed by polymerase activation at 95 °C for 2 min, and finally, 35 cycles of amplification at 95 °C for 15 s and 55 °C for 30 s. All samples were run in duplicate, including positive and no template controls.

Viral load was quantified in pharyngeal and nasal swabs as well as supernatant and cells from BAL for the animals housed at TNPRC using RT-qPCR. Probes targeting the nucleocapsid or envelope gene were used to quantify both genomic and subgenomic RNA of SARS-CoV-2. A Zymo Quick RNA Viral Kit (#R1035 or #D7003), Zymo, USA) was used to isolate RNA according to manufacturer’s protocol and as previously described (17 (link)). RNA was analyzed using a QuantStudio 6 (Thermo Scientific, USA) and TaqPath master mix (Thermo Scientific, USA) as previously described (17 (link)).

Nasopharyngeal swab were collected by a trained clinician with a flexible nylon swab that was inserted into the patient’s nostrils to reach the posterior nasopharynx. The swab was left in place for several seconds and slowly removed while rotating. The swab was then placed in 2 mL of sterile viral transport medium. Swabs from both nostrils were deposited in a single viral transport tube, taken to a clinical laboratory, and processed immediately.
Total nucleic acid was extracted from 300 μL of viral transport medium from the NPSs or 300 μL of whole saliva using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit (Thermo Fisher Scientific, Waltham, MA, United States) and eluted into 50 μL of elution buffer.
For SARS-CoV-2 RNA detection, 5 μL of RNA template was tested using TaqPath master mix (Thermo Fisher Scientific, Waltham, MA, USA). All tests were run on a Thermo Fisher ABI QuantStudio 5 real-time thermal cycler (Thermo Fisher Scientific, Waltham, MA, United States). Samples were selected for inclusion in this study based on viral Ct < 30.

The growth of the N. gonorrhoeae cells in different incubators was checked by analyzing the presence of the N. gonorrhoeae opa gene through real-time PCR. The opa gene and the primers used in PCR of this work were commonly found in literatures to identify N. gonorrhoeae. The sequence of the primers and probes were given in Table 2. The TaqPath master mix (Thermo Fisher Scientific, Waltham, MA) containing a primer and probe set targeting the N. gonorrhoeae opa gene was added with 2 μL of retrieved cell suspension from different incubators after 15 hr of incubation [17 (link)]. Cells collected before being placed in the incubators were used as a 0 hr control. The PCR amplification was performed in a real-time thermal cycler (CFX-96, Bio-Rad, Hercules, CA), with 5 minutes of initial activation and 50 cycles of PCR with 10 seconds of denaturation at 95 °C and 20 seconds of annealing and extension at 60 °C.

Isolated RNA was analyzed in a QuantStudio 6 (Thermo Scientific, USA) using TaqPath master mix (Thermo Scientific, USA) and appropriate primers/probes (S2 Table) with the following program: 25°C for 2 minutes, 50°C for 15 minutes, 95°C for 2 minutes followed by 40 cycles of 95°C for 3 seconds and 60°C for 30 seconds. Signals were compared to a standard curve generated using in vitro transcribed RNA of each sequence diluted from 10⁸ down to 10 copies. Positive controls consisted of SARS-CoV-2 infected VeroE6 cell lysate. Viral copies per swab were calculated by multiplying mean copies per well by amount in the total swab extract, while viral copies in tissue were calculated per μg of RNA extracted from each tissue.

Viral RNA in collected aerosol samples was quantified using RT-qPCR targeting the nucleocapsid (genomic) of SARS- CoV-2. RNA was isolated from aerosol samples using a Zymo Quick RNA Viral Kit (#R1035, Zymo, Irvine, CA, USA), per manufacturer′s instructions. RNA was eluted in RNAse-free water and was extracted using 100 μL of sample. Isolated RNA was analyzed in a QuantStudio 6 (Thermo Scientific, USA) using TaqPath master mix (Thermo Scientific, USA) and appropriate primers/probes [24 (link)] with the following program: 25 °C for 2 min, 50 °C for 15 min, and 95 °C for 2 min followed by 40 cycles of 95 °C for 3 s and 60 °C for 30 s. Signals were compared to a standard curve generated using in vitro transcribed RNA of each sequence diluted from 10⁸ down to 10 copies. Positive controls consisted of SARS-CoV-2-infected VeroE6 cell lysate. Viral copies per sample were calculated by multiplying mean copies per well by amount in the total sample extract.