Detection of infectious canine hepatitis virus by TaqMan real-time PCR method

Wang, Hye-young; Choi, Jae-yong; Lee, Mi-jin; Park, Jin-ho; Cho, Mae-Rim; Han, Jae-cheol; Choi, Kyoung-seong; Chae, Joon-seok

Korean Journal of Veterinary Research 2004;44(4):655-662.

Hye-young Wang, Jae-yong Choi, Mi-jin Lee, Jin-ho Park, Mae-Rim Cho, Jae-cheol Han, Kyoung-seong Choi, Joon-seok Chae

Bio-Safety Research Institute and College of Veterinary Medicine, Chonbuk National University

TaqMan 실시간 PCR법에 의한 개 전염성 간염 바이러스의 검출

왕혜영, 최재용, 이미진, 박진호, 조매림, 한재철, 최경성, 채준석

전북대학교 수의과대학 생체안전성 연구소

Abstract

The aim of this work was the validation of a rapid real-time PCR assay based on TaqMan technology for the unequivocal identification of infectious canine hepatitis (ICH) virus, to be used directly on DNA purified from blood specimens. A real-time PCR system targeting at the E3 ORFA gene sequence of canine adenovirus type 1 was optimized and validated through comparative analysis of samples using conventional PCR system. The real-time PCR assay based on TaqMan technology could disclose 23 (37.7%) out of 61 samples as PCR positive. In contrast, 18 (29.5%) samples were found PCR positive when conventional PCR was applied on these samples. The use of the ABI Prism 7700 sequence detection system allowed the efficient determination of the amplified product accumulation through a fluorogenic probe. The entire real-time TaqMan PCR assay, including DNA extraction, amplification, and detection could be completed within 3 hours. The detection method of real-time TaqMan PCR assay was 1,000 times more sensitive than conventional PCR. Real-time TaqMan probe and primer set developed and optimized in this study is a sensitive, rapid and accurate method for detection of ICH virus and can be effective screening tool for the detection of ICH in a diagnostic laboratory routines.

Key Words: infectious canine hepatitis, canine adenovirus, E3 ORFA gene, real-time PCR