Abstract
Avian influenza viruses (AIV) are negative sense RNA viruses posing a major threat to the poultry industry worldwide, with the potential to spread to mammals, including humans; hence, an accurate and rapid AIV diagnosis is essential. To date AIV detection relies on molecular methods, mainly RT-qPCR directed against AIV M gene segment. The evolution of AIV represents a relevant issue in diagnostic RT-qPCR due to possible mispriming and/or probe-binding failures resulting in false negative results. Consequently, RT-qPCR for AIV detection should be periodically re-assessed both in silico and in vitro. To this end, a specific workflow was developed to evaluate in silico the complementarity of primers and probes of four published RT-qPCR protocols to their target regions. The four assays and one commercially available kit for AIV detection were evaluated both for their analytical sensitivity using eight different viral dilution panels and for their diagnostic performances against clinical specimens of known infectious status. Differences were observed among the tests under evaluation, both in terms of analytical sensitivity and of diagnostic performances. This finding confirms the importance of continuously monitoring the primers and probes complementarity to their binding regions.
Highlights
Avian influenza viruses (AIV) are negative sense RNA viruses posing a major threat to the poultry industry worldwide, with the potential to spread to mammals, including humans; an accurate and rapid AIV diagnosis is essential
The dataset obtained for protocol 4 contains only one major cluster, showing 100% identity within the primers and probes binding regions and accounting for 3918 AIV M gene segment sequences (95.8%)
Avian influenza viruses exhibit a significant degree of genetic variability; this might lead to diagnostic failures of molecular tests when applied to mutated or new emerging viruses, meaning that a constant monitoring of the efficacy of molecular protocols available is uttermost necessary even when directed towards parts of the viral genome conventionally considered more stable
Summary
Avian influenza viruses (AIV) are negative sense RNA viruses posing a major threat to the poultry industry worldwide, with the potential to spread to mammals, including humans; an accurate and rapid AIV diagnosis is essential. Differences were observed among the tests under evaluation, both in terms of analytical sensitivity and of diagnostic performances This finding confirms the importance of continuously monitoring the primers and probes complementarity to their binding regions. The approach to AIV diagnosis using molecular methods adopted in most laboratories has been based on the initial generic detection of AIV in clinical specimens, primarily by targeting the matrix (M) gene segment, followed by specific RT-qPCR tests for H5 and H7 subtype viruses[1]. AIV, with its single-stranded negative-sense RNA genome, arranged into eight genomic segments, shows an intrinsic genetic instability[2] This is mainly due to the error-prone nature of the virus replication machinery and to re-assortment during infection of a single host cell with two or more distinct AIV types, resulting in considerable genetic heterogeneity and evolutionary diversity[2]. We compared the analytical sensitivity and the diagnostic performances of four published protocols for AIV M gene segment detection[6,8,9,10] and a commercially available diagnostic kit
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