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Evaluation of a pseudotype virus neutralisation test for measurement of equine influenza virus (EIV) antibody responses induced by vaccination and experimental infection

Abstract

Study design: Assay optimisation. Methods: Four sgRNA between 20 and 24 nucleotides in length complementary to different regions of the segment 7 (M gene) of H3N8 EIV, were designed using the CRISPRscan software, based in CAS12a predicted guides. Complete M gene of EIV H3N8 Florida clade 1 (FC1) and Florida clade 2 (FC2) representative strains were amplified by one step RT-PCR. The CRISPR-Cas12 detection step was carried out by incubating the PCR products with the CRISPR complex at 37ºC, and fluorescence was measured for up to 60 minutes every 5 minutes (ssDNA FQ substrate λex = 485 nm; λem = 535 nm). Results: All the designed sgRNA were able to detect the EIV H3N8 of FC1 while 2 of them detected FC2 representative strains, between 5 and 20 minutes. Main limitations: Limit of detection was not established and clinical samples were not assayed. Conclusions: CRISPR-Cas12a technology seems to be a promising system for detection of equine influenza virus, offering a convenient tool for low-resource laboratories. Ethical animal research: Not applicable.

Dates and versions

hal-03385023 , version 1 (21-10-2021)

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Rebecca Kinsley, Stéphane Pronost, Manuelle de Bock, Nigel Temperton, Janet M. Daly, et al.. Evaluation of a pseudotype virus neutralisation test for measurement of equine influenza virus (EIV) antibody responses induced by vaccination and experimental infection. 11th International Equine Infectious Diseases Conference, Sep 2021, Online, France. pp.76, ⟨10.1111/evj.118_13495⟩. ⟨hal-03385023⟩
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