基于牛结节性皮肤病病毒ORF61基因荧光定量检测方法的建立

doi:10.11843/j.issn.0366-6964.2024.04.041

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (4): 1800-1809.doi: 10.11843/j.issn.0366-6964.2024.04.041

基于牛结节性皮肤病病毒ORF61基因荧光定量检测方法的建立

马春玲^1,2, 任善会^1*, 杨雪¹, 蔺玉刚³, 李积雲⁴, 王相伟¹, 殷相平¹, 孙跃峰¹, 万学瑞^2*, 陈豪泰^1*

1. 中国农业科学院兰州兽医研究所, 兰州 730046;
2. 甘肃农业大学, 兰州 730070;
3. 塔里木大学, 阿拉尔 843300;
4. 青海省科学技术信息研究所, 西宁 810003

收稿日期:2023-07-21 出版日期:2024-04-23 发布日期:2024-04-26
通讯作者: 任善会,主要从事草食动物病毒致病机制相关研究,E-mail:renshanhui@caas.cn;万学瑞,主要从事病原微生物分子生物学研究,E-mail:wanxr@gsau.edu.cn;陈豪泰,主要从事草食动物病毒致病机制相关研究,E-mail:chenhaotai@caas.cn
作者简介:马春玲(1994-)，女，回族，甘肃康乐人，硕士生，主要从事草食动物病毒病感染致病机制研究，E-mail：1721105420@qq.com
基金资助:
国家自然科学基金(32302850)；甘肃省科技计划资助(22JR5RA035)；甘肃省科技重大专项(22ZD6NA001)；中国农业科学院兰州兽医研究所基本科研业务费(1610312021008)

Establishment and Application of Fluorescence Quantitative PCR Detection Method based on Lumpy Skin Disease Virus ORF61 Gene

MA Chunling^1,2, REN Shanhui^1*, YANG Xue¹, LIN Yugang³, LI Jiyun⁴, WANG Xiangwei¹, YIN Xiangping¹, SUN Yuefeng¹, WAN Xuerui^2*, CHEN Haotai^1*

1. Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
2. Gansu Agricultural University, Lanzhou 730070, China;
3. Tarim University, Alar 843300, China;
4. Qinghai Academy of Science and Technology, Xining 810003, China

Received:2023-07-21 Online:2024-04-23 Published:2024-04-26

摘要/Abstract

摘要： 基于国内流行的牛结节性皮肤病病毒(lumpy skin disease virus, LSDV)的基因组序列，建立了靶向ORF61基因的MGB探针荧光定量PCR检测方法。本研究基于LSDV基因序列设计出带有MGB探针的特异性荧光定量PCR引物，并进行特异性和灵敏性筛选验证。最终，筛选得到靶向于LSDV ORF61基因的一对qPCR引物；利用pCAGGS-ORF61真核表达质粒，获得标准曲线y=-3.287 5x+47.87，线性相关系数R²=0.998 6，扩增效率达101%；荧光定量PCR特异性、重复性和灵敏性试验结果表明，该引物的特异性高、重复性良好和灵敏度高等优点；该引物的最低检测限为6.71拷贝·μL^-1，各批次内与批次间重复性结果的变异系数均小于2%。以上结果表明，作者建立特异性LSDV的荧光定量PCR检测方法，为LSD预防和控制提供有效的检测手段。

关键词: 牛结节性皮肤病病毒, ORF61, MGB探针, 荧光定量PCR

Abstract: Based on the genomic sequence of a prevalent Lumpy skin disease virus (LSDV) in China, we established an MGB probe fluorescence quantitative PCR method for detecting the ORF61 gene. In this study, a specific fluorescent quantitative PCR primer with an MGB probe was designed based on the LSDV gene sequence, and the specificity and sensitivity of this primer pair were screened and verified. Finally, we screened a pair of qPCR primers targeting the LSDV ORF61 gene. Using pCAGGS-ORF61, a eukaryotic expression plasmid, the standard curve y=-3.287 5x+47.87 was obtained, the linear correlation coefficient was R²=0.998 6, and the amplification efficiency reached 101%. The results of the specificity test, repeatability test, and sensitivity test showed that this fluorescent quantitative PCR primer pair had the advantages of high specificity, good repeatability, and high sensitivity. The minimum detection limit of this primer pair was 6.71 copies·μL^-1, and the coefficient of variation of intra-batch and inter-batch repeatability results was less than 2%. The above results indicate that we have established a fluorescence quantitative PCR method for the specific detection of LSDV, providing an effective detection method for preventing and controlling LSD.

Key words: lumpy skin disease virus, ORF61, MGB probe, fluorescence quantitative PCR

中图分类号:

S852、659.1

马春玲, 任善会, 杨雪, 蔺玉刚, 李积雲, 王相伟, 殷相平, 孙跃峰, 万学瑞, 陈豪泰. 基于牛结节性皮肤病病毒ORF61基因荧光定量检测方法的建立[J]. 畜牧兽医学报, 2024, 55(4): 1800-1809.

MA Chunling, REN Shanhui, YANG Xue, LIN Yugang, LI Jiyun, WANG Xiangwei, YIN Xiangping, SUN Yuefeng, WAN Xuerui, CHEN Haotai. Establishment and Application of Fluorescence Quantitative PCR Detection Method based on Lumpy Skin Disease Virus ORF61 Gene[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(4): 1800-1809.

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基于牛结节性皮肤病病毒ORF61基因荧光定量检测方法的建立

Establishment and Application of Fluorescence Quantitative PCR Detection Method based on Lumpy Skin Disease Virus ORF61 Gene

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