Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 May 21;10(1):8441.
doi: 10.1038/s41598-020-64003-6.

Detection of Avian Influenza Virus: A Comparative Study of the in Silico and in Vitro Performances of Current RT-qPCR Assays

Free PMC article

Detection of Avian Influenza Virus: A Comparative Study of the in Silico and in Vitro Performances of Current RT-qPCR Assays

Andrea Laconi et al. Sci Rep. .
Free PMC article


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.

Conflict of interest statement

The authors declare no competing interests.


Figure 1
Figure 1
Schematic representation of the workflow used to evaluate the genetic diversity amongst primers and probes binding regions.
Figure 2
Figure 2
Entropy plots. (A) The nucleotide variation amongst the 99 bp fragment of the AIV M gene segment (positions 25–124) amplified by protocols 1, 2 and 3 shown as entropy. (B) The nucleotide variation amongst the 182 bp fragment of the AIV M gene segment (position 77–259) amplified by protocol 4 shown as entropy. The height of each column is proportional to the nucleotide variation at the given position. In red the primers and probes binding regions.

Similar articles

See all similar articles


    1. Avian Influenza (Infection with avian influenza viruses). OIE Terr. Man. 2015 - Chapter 2.3.4 (2015).
    1. Saurez, D. E. Common aspects of animal influenza. Anim. Influ. 3–29 (2017).
    1. Monne I, et al. Development and validation of a one-step real-time PCR assay for simultaneous detection of subtype H5, H7, and H9 avian influenza viruses. J. Clin. Microbiol. 2008;46:1769–1773. doi: 10.1128/JCM.02204-07. - DOI - PMC - PubMed
    1. Slomka MJ, et al. Identification of Sensitive and Specific Avian Influenza Polymerase Chain Reaction Methods Through Blind Ring Trials Organized in the European Union. Avian Dis. 2007;51:227–234. doi: 10.1637/7674-063006R1.1. - DOI - PubMed
    1. Spackman E, Ip HS, Suarez DL, Slemons R, Stallknecht D. Analytical validation of a real-time RT-PCR test for Pan-American lineage H7 subtype avian influenza viruses. J. Vet. Diagnostic Investig. 2008;20:612–616. doi: 10.1177/104063870802000512. - DOI - PubMed

LinkOut - more resources