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. 2022 Sep;69(5):e2261-e2267.
doi: 10.1111/tbed.14565. Epub 2022 Apr 28.

Novel duplex RT-qPCR for animal rabies surveillance

Guilherme Augusto Minozzo  1   2 , Thaila Francini Corona  1 , Elaine Cristina Rodrigues da Cruz  1 , Wagner Antonio Chiba de Castro  3 , Louise Bach Kmetiuk  4 , Andrea Pires Dos Santos  4 , Alexander Welker Biondo  5 , Irina Nastassja Riediger  1
Affiliations

Novel duplex RT-qPCR for animal rabies surveillance

Guilherme Augusto Minozzo et al. Transbound Emerg Dis. 2022 Sep.

Abstract

Rabies is a lethal zoonosis affecting mammals worldwide. Diagnosis of rabies follows international standard protocols, primarily relying on direct immunofluorescence (DI) followed by mouse inoculation test (MIT). WHO recommends molecular biology techniques such as RT-qPCR for replacing MIT to diagnose rabies in animal samples. Recently, a real-time PCR protocol that detects all rabies virus variants identified worldwide was validated. This assay is a pan-Lyssavirus TaqMan quantitative RT-PCR called LN34. A modified LN34 assay protocol was tested at the Paraná State Reference Laboratory (Lacen/PR) using animal samples previously tested by DI and MIT, the gold standard (GS). This method has been changed to a RT-qPCR duplex format to better fit the diagnostic routine. The new assay was called duplex LN34 and β-actin RT-qPCR. All the 88 samples evaluated using the GS test, modified pan-Lyssavirus TaqMan RT-qPCR and duplex LN34 and β-actin RT-qPCR showed 100% agreement with each other. This novel duplex RT-qPCR protocol has shown adequate diagnostic performance and may be used in research and surveillance purposes, replacing the standard MIT and ending mice use for rabies diagnosis.

Keywords: Cell culture; diagnosis; molecular techniques; rabies virus (RABV); real-time RT-qPCR assays; zoonoses.

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References

REFERENCES

    1. Appler, K., Brunt, S., Jarvis, J. A., & Davis, A. D. (2019). Clarifying indeterminate results on the rabies direct fluorescent antibody test using real-time reverse transcriptase polymerase chain reaction. Public Health Reports, 134, 57-62. https://doi.org/10.1177/0033354918810776
    1. Begeman, L., GeurtsvanKessel, C., Finke, S., Freuling, C. M., Koopmans, M., Müller, T., Ruigrok, T. J. H., & Kuiken, T. (2018). Comparative pathogenesis of rabies in bats and carnivores, and implications for spillover to humans. Lancet Infectious Diseases, 18, e147-e159. https://doi.org/10.1016/S1473-3099(17)30574-1
    1. Castilho, J. G., Achkar, S. M., de Novaes Oliveira, R., Mori, E., Carnieli, P. J., & Macedo, C. I. (2018). Analysis of rabies diagnosis in dogs and cats in the state of São Paulo, Brazil. Archives of Virology, 163, 2369-2376. https://doi.org/10.1007/s00705-018-3829-3
    1. Corona, T. F., Böger, B., da Rocha, T. C., Svoboda, W. K., & Gomes, E. C. (2018). Comparative analysis of mouse inoculation test and virus isolation in cell culture for rabies diagnosis in animals of Parana, Brazil. Revista Da Sociedade Brasileira De Medicina Tropical, 51, 39-43. https://doi.org/10.1590/0037-8682-0303-2017
    1. Dacheux, L., Larrous, F., Lavenir, R., Lepelletier, A., Faouzi, A., Troupin, C., Nourlil, J., Buchy, P., & Bourhy, H. (2016). Dual combined real-time reverse transcription polymerase chain reaction assay for the diagnosis of lyssavirus infection. PLOS Neglected Tropical Diseases, 10, e0004812. https://doi.org/10.1371/journal.pntd.0004812