Multiplex RT-qPCR  for RSV A and B Typing

Albert Campoy; Maria Iglesias; Sonia Vázquez-Morón; Francisco Pozo; Inma Casas

Apr 04, 2025

Multiplex RT-qPCR for RSV A and B Typing

DOI

dx.doi.org/10.17504/protocols.io.n2bvjdy7bvk5/v1

Albert Campoy¹,
Maria Iglesias¹,
Sonia Vázquez-Morón^1,2,
Francisco Pozo^1,2,
Inma Casas^1,2

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.

Albert Campoy

National Institute of Health Carlos III

DOI: https://dx.doi.org/10.17504/protocols.io.n2bvjdy7bvk5/v1

Protocol Citation: Albert Campoy, Maria Iglesias, Sonia Vázquez-Morón, Francisco Pozo, Inma 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

Keywords: typing of respiratory syncytial virus, respiratory syncytial virus, qpcr assay, qpcr assay for the detection, qpcr, rsv, multiplex rt, simultaneous identification of both target, virus, rt, assay for the detection, multiplex

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.

Troubleshooting

Materials

Primers:

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

Probes:

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

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

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

	Primer	Sequence (5'-3')
	RSAB1F	TTCTTGTTAGGTGTWGGATCTGCAAT
	RSAB1R	GTTACACCTGCATTRACACTRAATTC
	RP-F	GACTTCAGCATGGCGGTGT
	RP-R	GAGCGGCTGTCTCCACAAGT

	Probe	Sequence (5'-3')
	RSVA-FAM	TCTACTATCCACAAACAAGGCTGTAGTC
	RSVB-HEX	TTCCAACAATCTGCTGTTCTTCTGCTGG
	RP-TxRd	TTCTGACCTGAAGGCTCTGCGCG

Reagent	Use []	1x (ul)
H2O		5.25
2x PCR Master Mix		12.5
RSAB1F Primer	40 uM	0.5*
RSAB1A Primer	40 uM	0.5*
RP-F Primer	20 uM	0.5*
RP-R Primer	20 uM	0.5*
RSV A-FAM Probe	10 uM	0.25
RSV B-HEX Probe	10 uM	0.25
RP-TxRd	2.5 uM	0.25
SuperScript III RT/Platinum		0.5

A	B	C
52ºC	30 min
95ºC	2 min
95ºC	15 seg	50x
55ºC	1 min 30 seg	50x

Public workspaceMultiplex RT-qPCR for RSV A and B Typing

Multiplex RT-qPCR for RSV A and B Typing