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Development of TaqMan Quantitative PCR Assays for Duplex Detection of Dirofilaria immitis COI and Dog GAPDH from Infected Dog Blood
Korean J Clin Lab Sci 2019;51:64-70  
Published on March 31, 2019
Copyright © 2019 Korean Society for Clinical Laboratory Science.

In Young Oh1, Kyung Tae Kim2, Sun-Yeong Gwon1,4, Ho Joong Sung1,3

1Department of Biomedical Laboratory Sciences, College of Health Science, Eulji University, Seongnam, Korea, 2ALPHAGENE Co., Ltd., Singu University, Business Incubation Center, Seongnam, Korea, 3BK21 Plus Program, Department of Senior Healthcare, Graduate School, Eulji University, Daejeon, Korea, 4Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju, Korea
Correspondence to: Ho Joong Sung Department of Biomedical Laboratory Sciences, College of Health Science, Eulji University, 553 Sanseongdea-ro, Soojeong-gu, Seongnam 13135, Korea
Tel: 82-31-740-7438, Fax: 82-31-740-7425 , E-mail: hjsung@eulji.ac.kr ORCID: https://orcid.org/0000-0002-5614-1350
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Dirofilaria immitis (D. immitis) is a filarial nematode that causes cardiopulmonary dirofilariasis in dogs. In the late stages of infection, infected dogs show one or more symptoms and advanced heart disorder with perivascular inflammation. To detect D. immitis specifically and efficiently in the early stages of infection, a duplex TaqMan qPCR assay was developed based on previous studies using primers and probes specialized to detect D. immitis cytochrome c oxidase subunit I (COI) and dog glyceraldehyde-3-phosphate dehydrogenase (GAPDH). As positive controls, plasmid DNAs were constructed from D. immitis COI or dog GAPDH and a TA-cloning vector. Simplex and duplex TaqMan qPCR assays were performed using the specific primers, probes, and genomic or plasmid DNA. The duplex reaction developed could detect D. immitis COI and dog GAPDH in the same sample simultaneously after optimization of the primer concentrations. The limit of detection was 25 copies for the simplex and duplex assays, and both showed good linearity, high sensitivity, and excellent PCR efficiency. The duplex assays for pathogen detection reduce the costs, labor, and time compared to simplex reactions. Therefore, the duplex TaqMan qPCR assay developed herein will allow efficient D. immitis detection and quantification from a large number of samples simultaneously.
Keywords : Dirofilaria immitis, Duplex detection, TaqMan quantitative real-time PCR


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