Primers and probes used for BRSV and SARS-CoV-2 quantification were based on the TaqMan® protocol. BRSV detection was performed by targeting the conserved region of the N gene, according to the protocol previously described [34 (link)]. The reactions were performed using 5 µL of RNA extracted, 10 µL of the AgPath-ID™ One-Step RT-PCR Reagents kit, 0.8 µL primers BRSV-F and BRSV-R (final concentration 0.4 µM), and probe BRSV-P (final concentration 0.2 µM). The detection of SARS-CoV-2 was performed by targeting the N2 region (nucleocapsid gene) [35 (link)]. It was used 5 μL of RNA, 10 µL of SuperScriptTM III Platinum TM kit qRT-qPCR One-Step (Invitrogen), and 1.5 µL of primer and probe mix for the N2 region (2019-nCoV RUO Kit, Integrated DNA Technologies, Coralville, IA, USA). Reactions were performed in the ABI PRISM 7500 (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions. The reaction cycle was programmed for 30 min at 50 °C, 10 min at 95 °C, and 40 cycles of 15 s at 95 °C, 30 s at 60 °C.
The quantification of BRSV and SARS-CoV-2 RNA was estimated using a standard curve, in which serial 10-fold dilutions were used, with copies ranging from 100 to 106 GC/µL and 101 to 105 GC/µL, respectively, of a fragment of double-stranded DNA (gBlock Gene Fragment, Integrated DNA Technologies) containing the sequence of the specific target amplification region. Samples were considered positive when at least two of the four wells (diluted and undiluted) had a cycle threshold (Ct) value lower than 40.
To preserve samples from cross-contamination, quality control of molecular procedures was ensured with the use of different rooms in each of the processing activities. Negative process controls (RNAse-free water) and no template controls were included in each RT-qPCR running.
The quantification of BRSV and SARS-CoV-2 RNA was estimated using a standard curve, in which serial 10-fold dilutions were used, with copies ranging from 100 to 106 GC/µL and 101 to 105 GC/µL, respectively, of a fragment of double-stranded DNA (gBlock Gene Fragment, Integrated DNA Technologies) containing the sequence of the specific target amplification region. Samples were considered positive when at least two of the four wells (diluted and undiluted) had a cycle threshold (Ct) value lower than 40.
To preserve samples from cross-contamination, quality control of molecular procedures was ensured with the use of different rooms in each of the processing activities. Negative process controls (RNAse-free water) and no template controls were included in each RT-qPCR running.
