In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses

Abstract

The ongoing evolution of microbial pathogens represents a significant issue in diagnostic PCR/qPCR. Many assays are burdened with false negativity due to mispriming and/or probe-binding failures. Therefore, PCR/qPCR assays used in the laboratory should be periodically re-assessed in silico on public sequences to evaluate the ability to detect actually circulating strains and to infer potentially escaping variants. In the work presented we re-assessed a RT-qPCR assay for the universal detection of influenza A (IA) viruses currently recommended by the European Union Reference Laboratory for Avian Influenza. To this end, the primers and probe sequences were challenged against more than 99,000 M-segment sequences in five data pools. To streamline this process, we developed a simple algorithm called the SequenceTracer designed for alignment stratification, compression, and personal sequence subset selection and also demonstrated its utility. The re-assessment confirmed the high inclusivity of the assay for the detection of avian, swine and human pandemic H1N1 IA viruses. On the other hand, the analysis identified human H3N2 strains with a critical probe-interfering mutation circulating since 2010, albeit with a significantly fluctuating proportion. Minor variations located in the forward and reverse primers identified in the avian and swine data were also considered.

Influenza: National Trends Using the National Inpatient Sample Database from 1993 to 2015

“…Recent advancements in the detection of influenza make surveillance of the virus feasible, quick, and cost-effective in the clinical setting [14][15][16]. Moreover, there has been recent interest in the public health sector in mining online data to effectively predict local changes in influenza flux to predict epidemics of the virus in an attempt to control outbreaks [17].…”

A universal RT-qPCR assay for “One Health” detection of influenza A viruses

“…Re-assessment of the original SVIP-MP assay [24], which included a more recent (2018) analysis of 99,353 downloaded IAV MP-sequences [43] confirmed the high sequence conservation of the primers, guided the need for a new fluorescent probe design. Based on a highly conserved stretch at nucleotide positions 218-237, a novel FAM-labelled and MGB-modified probe SVIP-MP_P2-MGB, was selected (Table 1).…”

“…The primers and the probe ( [43]. Briefly, the sequences were downloaded in five data pools (Table 2) and aligned with the MAFFT tool [49].…”

“…Many of these assays were designed and validated in the context of IAV detection in a single species group. Although the MP-segment is relatively conserved in the IAV genome, certainly when compared to the subtype-determining HA and NA genes, continuing genetic drift has contributed to considerable nucleotide variability [41][42][43]. Therefore, there is an ongoing risk that the unpredictable emergence of new IAV strains with critical mismatches in the primer and/or probe binding sequences may compromise the sensitivity of the RT-qPCR assay, therefore requiring assay modifications or replacement [23,36,44,45].…”

“…In the case of modification of the MP-segment RT-qPCR [24] based on the analyses of 99,353 of IAV MP-segment sequences available in the public sequence databases, together with its validation to fulfil this test criterion. Crucially, the modified assay design employed novel, more refined, in silico bioinformatics approaches [43].…”