Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR

Abstract

Background: Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses.

Objectives: Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity.

Study design: Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples.

Results: N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses.

Conclusions: An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy.

Keywords: Mismatches; Primer; Probe; RSV; RT-PCR; Real-time.

Fig. 1

Summary of samples and rRT-PCR sensitivity for two RSV epidemics in Kilifi, Coastal Kenya 2014–2016.

Fig. 2

Phylogenetic clustering of nucleotide sequences from viruses undetectable and detectable by multiplex rRT-PCR from Kilifi, Coastal Kenya 2014–16. (a) Maximum likelihood tree of 72 N gene (700 bp) sequences: tips are colored by rRT-PCR outcome, where red is rRT-PCR negative (n = 40) and black is rRT-PCR positive (n = 32). (b) Maximum likelihood tree of 66 G gene (905 bp) sequences. Tips are colored as in (a). Viruses undetected by rRT-PCR formed a cluster separate from the detectable viruses sampled in the same epidemic. N and G gene sequences are derived from the same samples (matching sequences). The two RT-PCR negative strains had an additional polymorphism as described in text.

Fig. 3

An alignment (5‘ – 3‘) of the primers and probe with a subset of N gene sequences from the rRT-PCR discrepant samples: dots indicate identity with primer or probe sequence. Mismatching nucleotides at positions 11, 14 and 17 of the probe are highlighted. Majority of the rRT-PCR negative RSV-B viruses had nucleotide changes at the 14th base. The grey circle shows the additional polymorphism at the 6th base for two PCR negative strains that clustered with rRT-PCR positive viruses.