Detecting BRSV RNA via RT-PCR

Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed on nasal secretion samples as previously described [13 (link)]. Briefly, nasal secretion sample aliquots were subjected to RNA extraction using RNAzol^® (Sigma-Aldrich, St. Louis, MO, USA) following manufacturers recommendations. Once extracted, the RNA templates were reverse transcribed and amplified with qScript™ XLT One-Step RT-qPCR ToughMix (Quantabio^®, Beverly, MA, USA) using BRSV specific primers and probes [13 (link)]. Each reaction (2.5 μL) was performed in a BioRad CFX96^® (Bio-Rad^®, Hercules, CA, USA) and results were analyzed by BioRad CFX manager^® (Bio-Rad^®, Hercules, CA, USA). The detection limit of the assay was established at 10¹ BRSV RNA copies/μL.