鲤疱疹病毒2型与3型双重PCR检测方法的建立与应用

陈永聪; 方勤美; 柯翎

doi:10.19303/j.issn.1008-0384.2023.06.003

鲤疱疹病毒2型与3型双重PCR检测方法的建立与应用

doi: 10.19303/j.issn.1008-0384.2023.06.003

福建省农业科学院生物技术研究所，福建　福州　350003

基金项目: 福建省科技计划公益类专项（2020R10270010、2021R1027008）

详细信息

作者简介:
陈永聪（1968 —），男，硕士，农艺师，研究方向：特色水产养殖及药物研发（E-mail：610388747@qq.com）

通讯作者:
柯翎（1979 —），男，硕士，副研究员，研究方向：水产种苗繁育研究（E-mail：13599388598@163.com）

中图分类号: S941.41
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出版历程
- 收稿日期: 2023-02-03
- 修回日期: 2023-04-11
- 网络出版日期: 2023-06-02
- 刊出日期: 2023-06-28

Dual PCR Detection of Type 2 and 3 Cyprinus Herpesvirus

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

摘要

摘要: 目的鲤疱疹病毒2型和3型对鲤科鱼类危害严重，本研究旨在建立快捷、高效且能同时检测2种鲤疱疹病毒的检测技术。方法根据鲤疱疹病毒 2 型和3型的DNA聚合酶基因保守序列，设计2对特异性引物，通过对双重PCR反应条件的优化，建立检测鲤疱疹病毒2型和3型的双重PCR方法；使用该方法对试验室保存的鲫和鲤病鱼组织样品进行检测。结果该方法能够分别对鲤疱疹病毒2型和鲤疱疹病毒3型各扩增出715 bp和456 bp的特异性条带，表现出良好的特异性；敏感性试验结果显示，该方法的检测极限值为100 copies·μL^-1，具有较高的灵敏性。使用该方法对本试验室保存的18份临床病料进行PCR检测并对PCR产物进行测序验证，结果显示CyHV-2和CyHV-3阳性的各有3份，其阳性率都为33.3%；分别对2份CyHV-2和CyHV-3阳性样品混合后进行检测，结果混合样品均为CyHV-2和CyHV-3双阳性，与常规检测方法得到的结果相同。结论本研究建立的双重PCR检测方法特异性强、灵敏度高，可用于CyHV-2和CyHV-3的快速检测和鉴别诊断。
- 鲤 /
- 疱疹病毒 /
- 双重PCR
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

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图 1 最佳退火温度 PCR 扩增

M：2000 DNA Marker；1～9 ：退火温度依次是52 、54、56、58、60、62、64、66、68 ℃。

Figure 1. PCR amplification under optimized annealing temperature

M: 2000 DNA Marker；1–9: annealing temperatures of 52 ℃, 54 ℃, 56 ℃, 58 ℃, 60 ℃, 62 ℃, 64 ℃, 66 ℃, and 68 ℃, respectively.

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图 2 最佳引物用量的双重 PCR 扩增

M：2000 DNA Marker；1～6：引物添加量分别为 0.1、0.2、0.3、0.4、0.5 和 0.6 μL。

Figure 2. PCR amplification with optimized primer concentration

M: 2000 DNA Marker; 1–6: amounts of added two pairs of primers 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 μL, respectively.

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图 3 双重PCR 敏感性试验结果

M：2000 DNA Marker；1：CyHV-2 和 CyHV-3混合质粒初始含量1×10¹¹ copies·μL⁻¹；2～12：以10倍比稀释1×10¹⁰ ～1×10⁰ copies·μL⁻¹混合质粒含量。

Figure 3. Sensitivity of Dual PCR Assay

M: 2000 DNA Marker; 1: mixed plasmid of CyHV-2 and CyHV-3 as template (initial concentration 1×10¹¹ copies·μL⁻¹) ; 2–12: diluted at 10 times ratio 1×10¹⁰–1×10⁰ copies·μL⁻¹.

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图 4 PCR 特异性试验结果

M：2000 DNA Marker；1：石斑鱼神经坏死病毒；2：石斑鱼虹彩病毒；3：大黄鱼虹彩病毒；4：蛙虹彩病毒；5：鳗疱疹病毒；6：CyHV-2 和 CyHV-3的混合样品；7：CyHV-2； 8：CyHV-3 。

Figure 4. Specificity of Dual PCR Assay

M: 2000 DNA Marker; 1: GNNV; 2: SGIV; 3: YCIV; 4: FV; 5: AHV; 6: mixed sample of CyHV-2 and CyHV-3; 7: CyHV-2; 8: CyHV-3.

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表 1 双重PCR引物序列

Table 1. Sequence of primer for Dual PCR Assay

引物名称 Primer name	引物序列（5′-3′） Primer sequence (5′-3′)	片段长度 Fragment length/bp
CyHV-2-F	CTGATTATTACGGAGAGTATGAC	715
CyHV-2-R	CTGTGAGACTTTTGTAGATTATTC	715
CyHV-3-F	CTATGCTGGAACTGGTGATC	456
CyHV-3-R	GAGAGATTCTGACGGTGAAG	456

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表 2 样品检测结果

Table 2. Detection on clinical samples by Dual PCR Assay

检测方法 Detection method	鱼种 Fish species	阳性样品数/样品数 Positive samples/Total samples
检测方法 Detection method	鱼种 Fish species	CyHV-2	CyHV-3
《金鱼造血器官坏死病毒检测方法》（GB/T 36194—2018） Detection method of goldfish haemotopoietic necrosis virus	金鱼	3/9	—
	混合样	2/2	—
《鲤疱疹病毒检测方法第一部分：锦鲤疱疹病毒》（SC/T 7212.1—2011） Detection methods of cyprinid herpesvirus(CyHV)Part 1: Koi herpevirus	锦鲤	—	3/9
	混合样	—	2/2
双重PCR方法 Double PCR detection	金鱼	3/9	—
	锦鲤	—	3/9
	混合样	2/2	2/2

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参考文献(14)

[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