Apr 04, 2025

Public workspaceMultiplex RT-qPCR for RSV A and B Typing

  • 1Laboratory of Reference and Research in Respiratory Viruses, National Centre for Microbiology, Instituto de Salud Carlos III, 28220 Majadahonda, Spain;
  • 2CIBER de Epidemiología y Salud Pública (CIBERESP), ISCIII, Madrid, Spain.
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Protocol CitationAlbert Campoy, Maria Iglesias, Sonia Vázquez-Morón, Francisco Pozo, Inmaculada Casas 2025. Multiplex RT-qPCR for RSV A and B Typing. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjdy7bvk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: April 03, 2025
Last Modified: April 04, 2025
Protocol Integer ID: 126087
Abstract
This SOP describes a multiplex RT-qPCR assay for the detection and typing of respiratory syncytial virus (RSV) into subgroups A and B, enabling simultaneous identification of both targets in a single reaction.
Materials
Materials
Primers:

PrimerSequence (5'-3')
RSAB1FTTCTTGTTAGGTGTWGGATCTGCAAT
RSAB1RGTTACACCTGCATTRACACTRAATTC
RP-FGACTTCAGCATGGCGGTGT
RP-RGAGCGGCTGTCTCCACAAGT

Probes:

ProbeSequence (5'-3')
RSVA-FAMTCTACTATCCACAAACAAGGCTGTAGTC
RSVB-HEXTTCCAACAATCTGCTGTTCTTCTGCTGG
RP-TxRdTTCTGACCTGAAGGCTCTGCGCG

Master Mix Preparation
Master Mix Preparation

ReagentUse []1x (ul)
H2O5.25
2x PCR Master Mix12.5
RSAB1F Primer40 uM0.5*
RSAB1A Primer
RP-F Primer20 uM0.5*
RP-R Primer
RSV A-FAM Probe10 uM0.25
RSV B-HEX Probe0.25
RP-TxRd2.5 uM0.25
SuperScript III RT/Platinum0.5
*This volume refers to a pull composed of the two primers

Cycling Conditions
Cycling Conditions

ABC
52ºC30 min
95ºC2 min
95ºC15 seg50x
55ºC1 min 30 seg