Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jan 5:12:1071288.
doi: 10.3389/fcimb.2022.1071288. eCollection 2022.

Rapid detection of influenza A viruses using a real-time reverse transcription recombinase-aided amplification assay

Huan Cui  1   2 , Cheng Zhang  1   3 , Fei Tu  1 , Kui Zhao  2 , Yunyi Kong  1 , Jie Pu  1 , Lei Zhang  1 , Zhaoliang Chen  3 , Yuanyuan Sun  3 , Yujie Wei  3 , Chuncai Liang  3 , Juxiang Liu  3 , Jun Liu  1 , Zhendong Guo  1
Affiliations

Rapid detection of influenza A viruses using a real-time reverse transcription recombinase-aided amplification assay

Huan Cui et al. Front Cell Infect Microbiol. .

Abstract

Introduction: Influenza A viruses (IAVs) are important pathogens of respiratory infections, causing not only seasonal influenza but also influenza pandemics and posing a global threat to public health. IAVs infection spreads rapidly, widely, and across species, causing huge losses, especially zoonotic IAVs infections that are more harmful. Fast and sensitive detection of IAVs is critical for controlling the spread of this disease.

Methods: Here, a real-time reverse transcription recombinase-aided amplification (real-time RT-RAA) assay targeting conserved positions in the matrix protein gene (M gene) of IAVs, is successfully established to detect IAVs. The assay can be completed within 20 min at 42°C.

Results: The sensitivity of the real-time RT-RAA assay was 142 copies per reaction at 95% probability, which was comparable to the sensitivity of the RT-qPCR assay. The specificity assay showed that the real-time RT-RAA assay was specific to IAVs, and there was no cross-reactivity with other important viruses. In addition, 100%concordance between the real-time RT-RAA and RT-qPCR assays was achieved after testing 120 clinical specimens.

Discussion: The results suggested that the real-time RT-RAA assay we developed was a specific, sensitive and reliable diagnostic tool for the rapid detection of IAVs.

Keywords: clinical diagnosis; influenza A viruses (IAVs); isothermal amplification; rapid diagnosis; reverse transcription recombinase-aided amplification (RT-RAA).

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Locations of the real-time RT-RAA primers and probe on the M gene sequence of different IAVs strains. Use dots to represent nucleotide residues that match the majority. The forward primer (F91-121) is shaded in green; the reverse primer (R197-230) is blue and the exo probe (p122-170) is red. The two T residues within p122-170 labeled with a fluorophore (FAM) and quencher (BHQ1) are marked with solid and hollow triangles, respectively. THF is marked with arrow.
Figure 2
Figure 2
Screening the optical primers for real-time RT-RAA detection. (A) Sketch map of primary primer screening. In the primer name, the numbers indicate the position within the M gene from H1N1 (GenBank accession no. KX879560.1). (B) Primary reverse primer screening results. The forward primer F2-31 was randomly selected to screen all five reverse primers. (C) Primary forward primer screening results. The picked reverse primer R202-231 was used to pick all five forward primers. (D) Sketch map of secondary primer screening. (E) Secondary reverse primer screening results. The picked forward primer F87-116 was applied to screen all seven reverse primers. (F) Secondary forward primer screening results. The picked reverse primer R197-230 was used to pick all seven forward primers.
Figure 3
Figure 3
Specificity of the real-time RT-RAA assay. Curves 1-14, nucleic acid templates corresponding to H1N1, H3N2, H5N1, H5N6, H7N9, H9N2, H11N3, IBV-V, IBV-Y, ICV, RSV-A, RSV-B, SARS-CoV-2 and negative control, respectively.
Figure 4
Figure 4
Sensitivity tests for IAVs. Curves 1–7, correspond to 105–100 copies and the negative control, respectively. (A) Results of the real-time RT-RAA assay. (B) Results of the RT-qPCR assay. (C) The detection limit of the real-time RT-RAA assay at 95% reliability (142 copies per reaction) is labeled with a rhomboid. (D) The detection limit of the RT-qPCR assay at 95% reliability (161 copies per reaction) is labeled with a rhomboid.
See this image and copyright information in PMC

References

    1. Akin L., Gözel M. G. (2020). Understanding dynamics of pandemics. Turk J. Med. Sci. 50 (Si-1), 515–519. doi: 10.3906/sag-2004-133 - DOI - PMC - PubMed
    1. Chauhan R. P., Gordon M. L. (2020). A systematic review analyzing the prevalence and circulation of influenza viruses in swine population worldwide. Pathogens 9 (5), 355. doi: 10.3390/pathogens9050355 - DOI - PMC - PubMed
    1. Chauhan R. P., Gordon M. L. (2022). An overview of influenza a virus genes, protein functions, and replication cycle highlighting important updates. Virus Genes 58 (4), 255–269. doi: 10.1007/s11262-022-01904-w - DOI - PubMed
    1. Ciminski K., Pfaff F., Beer M., Schwemmle M. (2020). Bats reveal the true power of influenza a virus adaptability. PloS Pathog. 16 (4), e1008384. doi: 10.1371/journal.ppat.1008384 - DOI - PMC - PubMed
    1. Duan X., Ma W., Jiao Z., Tian Y., Ismail R. G., Zhou T., et al. (2022). Reverse transcription-recombinase-aided amplification and CRISPR/Cas12a-based visual detection of maize chlorotic mottle virus. Phytopathol. Res. 4 (1), 23. doi: 10.1186/s42483-022-00128-y - DOI - PMC - PubMed

MeSH terms