Development of a Duplex Real-time Fluorescence PCR for Simultaneous Detection of the Carp Edema Virus and Koi Herpesvirus
【Abstract】To establish a highly efficient diagnostic method for simultaneous detection of the carp edema virus (CEV) and koi herpesvirus (KHV), one pair of specific primers and a fluorescein FAM-labeled probe designed by the Center for Environment, Fisheries and Aquaculture Science according to conserved sequences of the P4a gene of the CEV were optimized, and one pair of specific primers and a VIC-labeled probe were designed on the basis of conserved sequences of the ORF7 of KHV. The duplex real-time polymerase chain reaction (PCR) was developed and optimized to detect the CEV and KHV in one reaction simultaneously. Results showed that the specificity of this PCR was high and there was no cross-reaction with other aquatic viruses. The limit of detection of this PCR was 15 copies/μL if two pre-mixed plasmids were present. The coefficient of variation was < 3% for intra-assay and inter-assay. It was shown that this duplex real-time PCR was a sensitive, specific and simple method for detection of the CEV and KHV which could be used as a rapid detection method.
【Keywords】 Carp edema virus (CEV) ; Koi herpesvirus (KHV) ; Duplex real-time fluorescence PCR;
(Translated by BAI XD)
 Xu L, Wang X, Zhang W, Pan Y, Jing H, Wang J, Wang Z, Chao H, Wang P, Na L, Jiang Y. Testing and identification of carp edema virus disease in cultured koi [J]. China Animal Husbandry & Veterinary Medicine, 2017, 44 (2): 613–618.
 Rakus K L, Ouyanq P, Boutier M, Ronsmans M, Reschner A, Vancsok C, Jazowiecka-Rakus J, Vanderplasschen A. Cyprinid herpesvirus 3: an interesting virus forapplied and fundamental research [J]. Vet Res, 2013, (44): 85.
 Xu L, Zhang W, Wang Z, Li X, Li Q, Wang L, Pan Y, Jiang Y. Preliminary survey and study of carp edema virus disease and koi herpesvirus disease Yellow River beach area in Henan [J]. Scientific Fish Farming, 2017, (9): 59–61.
 Wen Z, Liu Y, Tang S, Wang F, Wang J, Shi X, Yu L, Zheng X, He J, Lan W, Jia P, Liu H. Identification and genotyping of carp edema virus in Yunnan koi farm [J]. Chinese Journal of Virology, 2017, 33 (6): 1–9.
 Miwa S, Ito T, Sano M. Morphogenesis of koi herpesvirus observed by electron microscopy [J]. J Fish Dis, 2007, 30 (12): 715–722.
 Miyazaki T, Kuzuya Y, Yasumoto S, Yasuda M, Kobayashi T. Histopathologlical an ultrastructural features of koi herpesvirus (KHV)-infected carp Cyprinus carpio, and the morphology and morphogenesis of KHV [J]. Dis Aquat Org, 2008, 80 (1): 1–11.
 Kishio H, Ogawa K eds. translated by Ren X. New atlas of fish diseases [M]. Beijing: Chinese Agricultural University Press, 2007.
 Zheng S, Wang Q, Li Y, Zeng W, Wang Y, Liu C, Liang H, Shi C. Advances in carp herpesvirus III research [J]. Chinese Journal of Virology, 2016, 32 (1): 108–120.
 Wang X, Xu L, Wang Z, Wang J, Cao H, Zhang W, Jia L. Detection and analysis of carp edema virus in farmed carp and koi [J]. China Animal Husbandry & Veterinary Medicine,2017, 44 (10): 213–218.
 Matras M, Borzym E, Stone D, Way K, Stachnik M, Maj-Paluch J, Palusińska M, Reichert M. Carp edema virus in Polish aquaculture-evidence of significant sequence divergence and a new lineage in common carp Cyprinuscarp (L. ) [J]. J Fish Dis. 2017, 40 (3): 319–325.
 Adamek M, Matras M, Junq-Schroers V, Teitqe F, Helinq M, Berqmann S M, Reichert M, Way K, Stone DM, Steinhagen D. Comparison of PCR method for the detection of genetic variants of carp edema virus [J]. Dis Aquat Organ, 2017, 126 (1): 75–81.
 SC/T7212.1-2011. Carp herpesvirus detection method part 1: koi herpesvirus [S]. Beijing: Chinese Agricultural Press, 2011.
 Xie Z X, Xie L J, Pang Y S, Lu Z F, Xie Z Q, Sun J H, Deng X W, Liu J B, Tang X F. Development of a multiplex real-time RT-PCR assay for detection of WSSV and IHHNV [J]. Acta Hydrobiol Sin, 2009, 33 (1): 22–27.
 Xie Z X, Xie L J, Liu J B, Pang Y S, Deng X E, Xie Z Q. Development of a multiplex real-Time RT-PCR assay for detection of avian influenz virus and newcastle disease virus [J]. Lett Biotechnol, 2008, 19 (3): 410–413.
 Swaminathan T R, Kumar R, Dharmaratnam A, Basheer V S, Sood N, Pradhan P K, Sanil N K, Vijayaqopal P, Jena J K. Emergence of carp edema virus (CEV) in cultured ornamental koi carp, Cyprinuscarpio koi inIndia [J]. J Gen Virol, 2016, 97 (12): 3392–3399.
 Oyamatsu T, Matoyama H, Yamamoto K, Fukuda H. A trial for the detection of carp edema virus by using polymerase chain reaction [J]. Suisanzoshoku, 1997, 45: 247–252.
 Gilad O, Yun S, Adkison M A, Way K, Willits N H, Bercovier H, Hedrick R P. Molecular comparison of isolates of an emerging fish pathogen, koi herpesvirus, and the effect of water temperature on mortality of experimentally infected koi [J]. J Gen Virol, 2003, 84 (Pt 10): 2661–2667.
 Tu C, Hsieh C Y, Chang S K, Su J F, Chen M M. Develpment of a cell line from the gill of koi and molecular characterization of KHV isolate in Taiwan [J]. Afr J Microbiol Res, 2013, 7 (28): 3665–3674.
 Gilad O, Yun S, Zaqmutt-Verqara F J, Leuteneqqer C M, Bercovier H, Hedrick R P. Concentrations of a Koi herpesvirus (KHV) in tissues of experimentally infected Cyprinus carpio koi as assessed by real-time TaqMan PCR [J]. Dis Aquat Org, 2004, 60 (3): 179–187.
 Meng Q, Lv W, Shan X, Chen D, Wang W, Qian A. Establishment and application of a TaQMan fluorescent quantitative PCR rapid detection method of koi herpesvirus [J]. Journal of Jilin Agricultural University, 2012, 34 (3): 339–342, 354.