Development of a direct duplex real-time PCR assay for rapid testing of domestic cat hepadnavirus

Maya Shofa; Akiho Ohkawa; Tamaki Okabayashi; Yasuyuki Kaneko; Saito, Akatsuki

doi:10.51094/jxiv.112

##article.authors##

Maya Shofa Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki
Akiho Ohkawa Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki
Tamaki Okabayashi Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki https://orcid.org/0000-0001-8614-6593
Yasuyuki Kaneko Veterinary Teaching Hospital, Faculty of Agriculture, University of Miyazaki
Saito, Akatsuki Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki

DOI:

https://doi.org/10.51094/jxiv.112

キーワード:

domestic cat hepadnavirus、 direct duplex real-time PCR assay、 rapid、 sensitive

抄録

Domestic cat hepadnavirus (DCH) is a novel hepadnavirus, first identified in 2018. The DCH is generally diagnosed using conventional polymerase chain reaction (PCR) assays, which include a time-consuming agarose gel electrophoresis. In this study, we developed a rapid, sensitive, and specific real-time PCR assay for the detection of the DCH genome. To streamline the procedure, our real-time PCR assay was carried out using blood samples, without deoxyribonucleic acid (DNA) extraction. A primers/probe set was designed based on the nucleotide sequences of the surface gene of the DCH strain Japan/KT116/2021 (Accession# LC668427), which we recently identified from a feline blood sample in Japan. To exclude the possibility that the PCR reaction was blocked by anticoagulants, we also used a primers/probe set for amplifying the housekeeping beta-actin gene. Direct duplex real-time PCR assay had a high sensitivity, with a limit of detection of 10 copies/μL for DCH. We successfully established a rapid and highly sensitive duplex real-time PCR assay for the detection and quantification of DCH. This direct duplex real-time PCR assay is a useful tool for DCH diagnosis and surveillance.

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引用文献

Aghazadeh, M., Shi, M., Barrs, V.R., McLuckie, A.J., Lindsay, S.A., Jameson, B., Hampson, B., Holmes, E.C., Beatty, J.A., 2018. A novel hepadnavirus identified in an immunocompromised domestic cat in Australia. Viruses 10, 269. https://doi.org/10.3390/v10050269

Anpuanandam, K., Selvarajah, G.T., Choy, M.M.K., Ng, S.W., Kumar, K., Ali, R.M., Rajendran, S.K., Ho, K.L., Tan, W.S., 2021. Molecular detection and characterisation of Domestic Cat Hepadnavirus (DCH) from blood and liver tissues of cats in Malaysia. BMC Veterinary Research 17, 9. https://doi.org/10.1186/s12917-020-02700-0

Diakoudi, G., Capozza, P., Lanave, G., Pellegrini, F., Di Martino, B., Elia, G., Decaro, N., Camero, M., Ghergo, P., Stasi, F., Cavalli, A., Tempesta, M., Barrs, V.R., Beatty, J., Bányai, K., Catella, C., Lucente, M.S., Buonavoglia, A., Fusco, G., Martella, V., 2022. A novel hepadnavirus in domestic dogs. Scientific Reports 12, 2864. https://doi.org/10.1038/S41598-022-06842-Z

Donahue, P.R., Hoover, E.A., Beltz, G.A., Riedel, N., Hirsch, V.M., Overbaugh, J., Mullins, J.I., 1988. Strong sequence conservation among horizontally transmissible, minimally pathogenic feline leukemia viruses. Journal ofVirology 62, 722–731. https://doi.org/10.1128/JVI.62.3.722-731.1988

Fruci, P., Di Profio, F., Palombieri, A., Massirio, I., Lanave, G., Diakoudi, G., Pellegrini, F., Marsilio, F., Martella, V., Di Martino, B., 2022. Detection of antibodies against domestic cat hepadnavirus using baculovirus-expressed core protein. Transboundary and Emerging Diseases n/a. https://doi.org/10.1111/tbed.14461

Hu, Y., 2016. Regulatory concern of polymerase chain reaction (PCR) carryover contamination. Polymerase Chain Reaction for Biomedical Applications. https://doi.org/10.5772/66294

Jeanes, E.C., Wegg, M.L., Mitchell, J.A., Priestnall, S.L., Fleming, L., Dawson, C., 2022. Comparison of the prevalence of domestic cat hepadnavirus in a population of cats with uveitis and in a healthy blood donor cat population in the United Kingdom. Veterinary Ophthalmology 25, 165–172. https://doi.org/10.1111/VOP.12956

Lacouture, S., Okura, M., Takamatsu, D., Corsaut, L., Gottschalk, M., 2020. Development of a mismatch amplification mutation assay to correctly serotype isolates of Streptococcus suis serotypes 1, 2, 1/2, and 14. Journal of Veterinary Diagnostic Investigation 32, 490–494. https://doi.org/10.1177/1040638720915869

Lanave, G., Capozza, P., Diakoudi, G., Catella, C., Catucci, L., Ghergo, P., Stasi, F., Barrs, V., Beatty, J., Decaro, N., Buonavoglia, C., Martella, V., Camero, M., 2019. Identification of hepadnavirus in the sera of cats. Scientific Reports 2019 9, 9, 10668. https://doi.org/10.1038/s41598-019-47175-8

Magnius, L., Mason, W.S., Taylor, J., Kann, M., Glebe, D., Dény, P., Sureau, C., Norder, H., Consortium, I.R., 2020. ICTV virus taxonomy profile: Hepadnaviridae. The Journal of General Virology 101, 571. https://doi.org/10.1099/JGV.0.001415

Overbaugh, J., Donahue, P.R., Quackenbush, S.L., Hoover, E.A., Mullins, J.I., 1988. Molecular cloning of a feline leukemia virus that induces fatal immunodeficiency disease in cats. Science 239, 906–910. https://doi.org/10.1126/science.2893454

Phillips, T.R., Talbott, R.L., Lamont, C., Muir, S., Lovelace, K., Elder, J.H., 1990. Comparison of two host cell range variants of feline immunodeficiency virus. Journal of Virology 64, 4605–4613. https://doi.org/10.1128/JVI.64.10.4605-4613.1990

Piewbang, C., Wardhani, S.W., Chaiyasak, S., Yostawonkul, J., Chai-In, P., Boonrungsiman, S., Kasantikul, T., Techangamsuwan, S., 2020. Insights into the genetic diversity, recombination, and systemic infections with evidence of intracellular maturation of hepadnavirus in cats. PLoS ONE 15, e0241212. https://doi.org/10.1371/journal.pone.0241212

Stadhouders, R., Pas, S.D., Anber, J., Voermans, J., Mes, T.H.M., Schutten, M., 2010. The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5′ nuclease assay. Journal of Molecular Diagnostics 12, 109–117. https://doi.org/10.2353/jmoldx.2010.090035

Takahashi, K., Kaneko, Y., Shibanai, A., Yamamoto, S., Katagiri, A., Osuga, T., Inoue, Y., Kuroda, K., Tanabe, M., Okabayashi, T., Naganobu, K., Minobe, I., Saito, A., 2022. Identification of domestic cat hepadnavirus from a cat blood sample in Japan. The Journal of Veterinary Medical Science 22–0010. https://doi.org/10.1292/jvms.22-0010

Talbott, R.L., Sparger, E.E., Lovelace, K.M., Fitch, W.M., Pedersen, N.C., Luciw, P.A., Elder, J.H., 1989. Nucleotide sequence and genomic organization of feline immunodeficiency virus. Proceedings of the National Academy of Sciences of the United States of America 86, 5743–5747. https://doi.org/10.1073/PNAS.86.15.5743

Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., Madden, T.L., 2012. Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13, 134. https://doi.org/10.1186/1471-2105-13-134