TNA extraction was performed using the MagNA Pure 96 extraction system (Roche, Switzerland) according to the manufacturer’s instructions, as we described previously (7 (link)). Briefly, 200 μL of each sample was mixed with MagNA Pure 96 external lysis buffer (Roche). After extraction, TNA was recovered in 50 μL of elution buffer and then kept at −80°C until use.
Before the preparation of a master mix for the reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay, 10× LAMP primer mix was prepared (see Table S3 in the supplemental material). The RT-LAMP reagent mixture (10 μL) contained 0.4 μL of nuclease-free water, 1 μL of 10× isothermal amplification buffer (NEB, USA), 0.6 μL of 100 mM MgSO4 (NEB), 1.4 μL of 10 mM dNTP solution mix, 1 μL of 10× LAMP primer mix, 0.4 μL of Bst 2.0 WarmStart DNA polymerase (8,000 U/mL) (NEB), 0.2 μL of WarmStart RTx reverse transcriptase (15,000 U/mL) (NEB), and 5 μL of TNA as the template. RT-LAMP reactions were performed at 60°C for 40 min for the COVID-19 nsp8 assay, at 62°C for 30 min for the COVID-19 N assay, and at 62°C for 40 min for the human RNase P assay.
Before the preparation of a master mix for the reverse transcription–loop-mediated isothermal amplification (RT-LAMP) assay, 10× LAMP primer mix was prepared (see Table S3 in the supplemental material). The RT-LAMP reagent mixture (10 μL) contained 0.4 μL of nuclease-free water, 1 μL of 10× isothermal amplification buffer (NEB, USA), 0.6 μL of 100 mM MgSO4 (NEB), 1.4 μL of 10 mM dNTP solution mix, 1 μL of 10× LAMP primer mix, 0.4 μL of Bst 2.0 WarmStart DNA polymerase (8,000 U/mL) (NEB), 0.2 μL of WarmStart RTx reverse transcriptase (15,000 U/mL) (NEB), and 5 μL of TNA as the template. RT-LAMP reactions were performed at 60°C for 40 min for the COVID-19 nsp8 assay, at 62°C for 30 min for the COVID-19 N assay, and at 62°C for 40 min for the human RNase P assay.
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