Development of a direct duplex real-time PCR assay for rapid testing of domestic cat hepadnavirus
##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
- Akatsuki Saito Department of Veterinary Science, Faculty of Agriculture, University of Miyazaki
DOI:
https://doi.org/10.51094/jxiv.112Keywords:
domestic cat hepadnavirus, direct duplex real-time PCR assay, rapid, sensitiveAbstract
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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Submitted: 2022-07-07 03:14:52 UTC
Published: 2022-07-07 08:55:49 UTC
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Maya Shofa
Akiho Ohkawa
Tamaki Okabayashi
Yasuyuki Kaneko
Akatsuki Saito
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