Dual PCR Detection of Type 2 and 3 <i>Cyprinus Herpesvirus</i>

CHEN Yongcong; FANG Qinmei; KE Ling

doi:10.19303/j.issn.1008-0384.2023.06.003

Volume 38 Issue 6

Jun. 2023

Turn off MathJax

Article Contents

Article Navigation > Fujian Journal of Agricultural Sciences > 2023 > 38(6): 652-656

CHEN Y C, FANG Q M, KE L. Dual PCR Detection of Type 2 and 3 Cyprinus Herpesvirus [J]. Fujian Journal of Agricultural Sciences，2023，38（6）：652−656 doi: 10.19303/j.issn.1008-0384.2023.06.003

Citation:

CHEN Y C, FANG Q M, KE L. Dual PCR Detection of Type 2 and 3 Cyprinus Herpesvirus [J]. Fujian Journal of Agricultural Sciences，2023，38（6）：652−656 doi: 10.19303/j.issn.1008-0384.2023.06.003

Citation:

PDF( 667 KB)

Dual PCR Detection of Type 2 and 3 Cyprinus Herpesvirus

doi: 10.19303/j.issn.1008-0384.2023.06.003

Biotechnology Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350003, China

Received Date: 2023-02-03
Rev Recd Date: 2023-04-11

Available Online: 2023-06-02

Publish Date: 2023-06-28

Abstract

Abstract

Objective A PCR method for simultaneously detecting Type 2 and Type 3 Cyprinid herpesvirus that cause serious diseases on Cyprinidae was developed and tested for clinical diagnosis. Methods Two pairs of specific primers were designed according to the conserved sequences of the DNA polymerase gene of the two types of virus, CyHV-2 and CyHV-3. PCR reaction conditions of the method were optimized. The assay was applied on the stored tissue samples of diseased Carassius auratus and Cyprinus carpio to verify validity of the methodology. Result The newly developed Dual PCR Assay amplified specific bands with the base numbers of 715 bp for CyHV-2 and 456 bp for CyHV-3. It exhibited high sensitivity with a detection limit of 100 copies·μL⁻¹. On the 18 clinical samples, 3 were found to be CyHV-2 and 3 CyHV-3 with a positive detection rate of 33.3%. Furthermore, the assay successfully identified the two type viruses in a mixed sample of CyHV-2 and CyHV-3 in a challenge test. Conclusion The Dual PCR Assay demonstrated high specificity and sensitivity in simultaneously detecting CyHV-2 and CyHV-3. It could be adequately applied for rapid diagnosis of the viral diseases on carps.
- Cyprinus carpio,
- Cyprinus herpesvirus,
- Double PCR

FullText(HTML)

References(14)

References

[1]	农业农村部渔业渔政管理局, 全国水产技术推广总站, 中国水产学会. 2021中国渔业统计年鉴[M]. 北京: 中国农业出版社, 2022: 25.
[2]	BALON E K. Origin and domestication of the wild carp, Cyprinus carpio: From Roman gourmets to the swimming flowers [J]. Aquaculture, 1995, 129(1/2/3/4): 3−48.
[3]	GOODWIN A E, SADLER J, MERRY G E, et al. Herpesviral haematopoietic necrosis virus (C_yHV-2) infection: Case studies from commercial goldfish farms [J]. Journal of Fish Diseases, 2009, 32(3): 271−278. doi: 10.1111/j.1365-2761.2008.00988.x
[4]	HEDRICK R P, GILAD O, YUN S, et al. A herpesvirus associated with mass mortality of juvenile and adult koi, a strain of common carp [J]. Journal of Aquatic Animal Health, 2000, 12(1): 44−57. doi: 10.1577/1548-8667(2000)012<0044:AHAWMM>2.0.CO;2
[5]	AOKI T, HIRONO I, KUROKAWA K, et al. Genome sequences of three koi herpesvirus isolates representing the expanding distribution of an emerging disease threatening koi and common carp worldwide [J]. Journal of Virology, 2007, 81(10): 5058−5065. doi: 10.1128/JVI.00146-07
[6]	BECKER J A, TWEEDIE A, RIMMER A, et al. Incursions of Cyprinid herpesvirus 2 in goldfish populations in Australia despite quarantine practices [J]. Aquaculture, 2014, 432: 53−59. doi: 10.1016/j.aquaculture.2014.04.020
[7]	李莉娟, 罗杨志, 刘学芹, 等. 金鱼鲤疱疹病毒Ⅱ型的分子诊断 [J]. 华中农业大学学报, 2013, 32(1):92−96. doi: 10.3969/j.issn.1000-2421.2013.01.018 LI L J, LUO Y Z, LIU X Q, et al. Molecular surveillance of Cyprinid herpesvirus 2 in goldfish cultured in China [J]. Journal of Huazhong Agricultural University, 2013, 32(1): 92−96.(in Chinese) doi: 10.3969/j.issn.1000-2421.2013.01.018
[8]	XU J R, BENTLY J, BECK L, et al. Analysis of koi herpesvirus latency in wild common carp and ornamental koi in Oregon, USA [J]. Journal of Virological Methods, 2013, 187(2): 372−379. doi: 10.1016/j.jviromet.2012.11.015
[9]	GOODWIN A E, KHOO L, LAPATRA S E, et al. Goldfish hematopoietic necrosis herpesvirus (cyprinid herpesvirus 2) in the USA: Molecular confirmation of isolates from diseased fish [J]. Journal of Aquatic Animal Health, 2006, 18(1): 11−18. doi: 10.1577/H05-007.1
[10]	WALTZEK T B, KUROBE T, GOODWIN A E, et al. Development of a polymerase chain reaction assay to detect cyprinid herpesvirus 2 in goldfish [J]. Journal of Aquatic Animal Health, 2009, 21(1): 60−67. doi: 10.1577/H08-045.1
[11]	BERCOVIER H, FISHMAN Y, NAHARY R, et al. Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis [J]. BMC Microbiology, 2005, 5: 13. doi: 10.1186/1471-2180-5-13
[12]	张文, 徐立蒲, 吕晓楠, 等. 鲤浮肿病毒和锦鲤疱疹病毒三重PCR检测方法的建立及初步应用 [J]. 检验检疫学刊, 2019, 29(1):1−5. ZHANG W, XU L P, LV X N, et al. Establishment and preliminary application of triplex PCR detection method for carp edema virus and koi herpesvirus [J]. Journal of Inspection and Quarantine, 2019, 29(1): 1−5.(in Chinese)
[13]	徐晔, 段宏安, 周毅, 等. 鲤科疱疹病毒2型TaqMan荧光定量PCR检测方法的建立 [J]. 中国预防兽医学报, 2014, 36(12):952−956. doi: 10.3969/j.issn.1008-0589.2014.12.09 XU Y, DUAN H A, ZHOU Y, et al. Establishment of real-time PCR assay for detection of Cyprinid herpesvirus 2 [J]. Chinese Journal of Preventive Veterinary Medicine, 2014, 36(12): 952−956.(in Chinese) doi: 10.3969/j.issn.1008-0589.2014.12.09
[14]	彭军辉, 安伟, 张明辉. 鲤疱疹病毒2型和3型三重PCR检测方法的建立与应用 [J]. 渔业科学进展, 2021, 42(6):158−164. doi: 10.19663/j.issn2095-9869.20201124002 PENG J H, AN W, ZHANG M H. Establishment and application of the triplex PCR method for simultaneous detection of cyprinid herpesvirus 2 and cyprinid herpesvirus 3 [J]. Progress in Fishery Sciences, 2021, 42(6): 158−164.(in Chinese) doi: 10.19663/j.issn2095-9869.20201124002