靶向山羊痘病毒GPCR基因SYBR Green荧光定量PCR检测方法的建立

doi:10.11843/j.issn.0366-6964.2024.10.047

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (10): 4779-4784.doi: 10.11843/j.issn.0366-6964.2024.10.047

• 研究简报 • 上一篇

靶向山羊痘病毒GPCR基因SYBR Green荧光定量PCR检测方法的建立

张红强¹(), 任善会²^,*(), 姚威³, 龚真莉², 杨雪¹, 马春玲², 尤婷¹, 张玉哲¹, 柳民意¹, 钱文洁¹, 李柳杨¹, 余志鹏¹, 孙跃峰², 陈豪泰², 樊江峰¹^,*()

1. 甘肃农业大学动物医学院, 兰州 730070
2. 中国农业科学院兰州兽医研究所, 兰州 730046
3. 重庆市万州区畜牧产业发展中心, 重庆 404100

收稿日期:2023-12-08 出版日期:2024-10-23 发布日期:2024-11-04
通讯作者: 任善会,樊江峰 E-mail:2306590936@qq.com;renshanhui@caas.cn;fanjf@gsau.edu.cn
作者简介:张红强(1993-), 男, 甘肃漳县人, 硕士生, 主要从事草食动物病毒病感染致病机制研究, E-mail: 2306590936@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);lanmu(bianhao);中国农业科学院兰州兽医研究所基本科研业务费(1610312021008)

The Establishment of a SYBR Green Fluorescence Quantitative PCR Detection Method by Targeting the G-protein Coupled Chemokine Receptor Gene of Goat Poxvirus

Hongqiang ZHANG¹(), Shanhui REN²^,*(), Wei YAO³, Zhenli GONG², Xue YANG¹, Chunling MA², Ting YOU¹, Yuzhe ZHANG¹, Minyi LIU¹, Wenjie QIAN¹, Liuyang LI¹, Zhipeng YU¹, Yuefeng SUN², Haotai CHEN², Jiangfeng FAN¹^,*()

1. College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, China
2. Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Scicences, Lanzhou 730046, China
3. Wanzhou Center for Animal Husbandry Industry Development, Chongqing 404100, China

Received:2023-12-08 Online:2024-10-23 Published:2024-11-04
Contact: Shanhui REN, Jiangfeng FAN E-mail:2306590936@qq.com;renshanhui@caas.cn;fanjf@gsau.edu.cn

摘要/Abstract

摘要：

基于山羊痘病毒(goat pox virus, GTPV)的基因组序列，建立靶向G蛋白偶联趋化因子受体(G-protein-coupled chemokine receptor，GPCR)基因的荧光定量PCR检测方法。基于GTPV全基因序列设计6对qPCR引物，并进行特异性和灵敏性的筛选和检测，最终筛选得到1对高特异性和灵敏性的荧光定量PCR引物；根据GTPV/AV41疫苗株GPCR基因序列，设计普通PCR引物，扩增GPCR基因，构建真核表达载体，建立荧光定量PCR标准曲线。结果表明：筛选得到1对靶向于GPCR基因的特异性qPCR引物；构建pCAGGS-GPCR真核表达质粒，建立标准曲线y=-3.5289x+49.07，线性相关系数R²=0.997 2，扩增效率为92.7%；验证性试验结果显示，该引物最低检测限为2.0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%，表明其具有特异性强、重复性好和灵敏度高的优点。建立了靶向山羊痘病毒GPCR基因的qPCR检测方法，为防控山羊痘提供了高效的检测手段。

关键词: 山羊痘病毒, GPCR, 荧光定量PCR

Abstract:

Based on the genomic sequence of the goat pox virus (GTPV), we have established a fluorescence quantitative PCR method targeting the G-protein-coupled chemokine receptor (GPCR) gene. Six pairs of specific qPCR primers were designed based on the whole genomic sequence of GTPV. The specificity and sensitivity of these six qPCR primers were screened and detected. A pair of qPCR primers with high specificity and sensitivity was finally screened and obtained. According to the GPCR gene sequence of the GTPV AV41 vaccine strain, we designed an ordinary PCR primer pair to amplify the GPCR gene to construct the eukaryotic expression vector, which was used to establish the standard curve. Results showed that A pair of specific qPCR primers targeting the GPCR gene was obtained. The eukaryotic expression plasmid named pCAGGS-GPCR was constructed. The standard curve of y=-3.5289x+49.07 was established, whose linear correlation coefficient is R²=0.997 2 and amplification efficiency is 92.7%. The results of the replication experiment suggested that the lowest detective limitation of this primer was 2.0 copies·μL^-1, and the coefficient variation of the reproducibility results within and between batches was 2%, indicating the advantages of reasonable specificity, repeatability, and sensitivity. We have successfully established a specific fluorescence quantitative PCR method targeting the GPCR gene of GTPV, which provides adequate detecting support for the veterinary clinical diagnosis and the prevention and control of goat poxvirus.

Key words: goat pox virus, GPCR, fluorescence quantitative PCR

中图分类号:

S852.654

张红强, 任善会, 姚威, 龚真莉, 杨雪, 马春玲, 尤婷, 张玉哲, 柳民意, 钱文洁, 李柳杨, 余志鹏, 孙跃峰, 陈豪泰, 樊江峰. 靶向山羊痘病毒GPCR基因SYBR Green荧光定量PCR检测方法的建立[J]. 畜牧兽医学报, 2024, 55(10): 4779-4784.

Hongqiang ZHANG, Shanhui REN, Wei YAO, Zhenli GONG, Xue YANG, Chunling MA, Ting YOU, Yuzhe ZHANG, Minyi LIU, Wenjie QIAN, Liuyang LI, Zhipeng YU, Yuefeng SUN, Haotai CHEN, Jiangfeng FAN. The Establishment of a SYBR Green Fluorescence Quantitative PCR Detection Method by Targeting the G-protein Coupled Chemokine Receptor Gene of Goat Poxvirus[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(10): 4779-4784.

图/表 5

表 1

表 2

图 1

图 2

图 3

参考文献 14

1	陆游. 牛结节性皮肤病病毒荧光定量PCR检测方法的建立与异源弱毒疫苗山羊痘病毒AV41株基因组分子标记的比较分析[D]. 扬州: 扬州大学, 2021.
	LU Y. Establishment of fluorescent quantitative PCR detectionmethod for lumpy skin disease virus and comparative analysis of genomic molecular markers with heterologous attenuated vaccine goatpox virus AV41[D]. Yangzhou: Yangzhou University, 2021. (in Chinese)
2	刘芳. 一起疑似羊痘病毒感染的实验室诊断及病毒分离鉴定[D]. 长春: 吉林大学, 2020.
	LIU F. These sheep were depressed and thin, whose temperature raised in the early stages[D]. Changchun: Jilin University, 2020. (in Chinese)
3	曾显成. 山羊痘病毒全基因组序列分析及抗病毒天然免疫应答机制研究[D]. 福州: 福建农林大学, 2015.
	ZENG X C. Complete genome sequence analysis of goatpox virus and the regulatory mechanism of host antiviral innate immunity[D]. Fuzhou: Fujian Agriculture And Forestry University, 2015. (in Chinese)
4	张莹辉, 姚文生, 康凯, 等. 山羊痘病毒检测方法研究进展[J]. 中国兽药杂志, 2018, 52 (3): 72- 77.
	ZHANG Y H , YAO W S , KANG K , et al. Research progress of detection methods of goat pox virus[J]. Chinese Journal of Veterinary Drug, 2018, 52 (3): 72- 77.
5	王宪军. 羊口疮病毒、小反刍兽疫病毒和羊痘病毒三重TaqMan荧光定量RT-PCR方法的建立及初步应用[D]. 成都: 西南民族大学, 2022.
	WANG X J. Establishment and application of triple TaqMan fluorescence quantitative RT-PCR method for sheep mouth sore virus, small ruminant disease virus and sheep pox virus[D]. Chengdu: Southwest Minzu University, 2022. (in Chinese)
6	岳帅. 羊传染性脓疱病毒及羊痘病毒双重荧光PCR方法的建立与应用[D]. 长春: 吉林农业大学, 2019.
	YUE S. Establishment and application of duplex real-time PCR Method for sheep contagious pustular dermatitis virus and sheep pox virus[D]. Changchun: Jilin Agricultural University, 2019. (in Chinese)
7	聂福平, 王昱, 黄秋华, 等. 牛结节性皮肤病病毒TaqMan-MGB荧光PCR检测方法的建立[J]. 中国兽医科学, 2017, 47 (9): 1129- 1134.
	NIE F P , WANG Y , HUANG Q H , et al. Establishment of TaqMan MGB fluorescent PCR detection method for bovine nodular skin disease virus[J]. Chinese Veterinary Science, 2017, 47 (9): 1129- 1134.
8	MAFIRAKUREVA P , SAIDI B , MBANGA J . Incidence and molecular characterisation of lumpy skin disease virus in Zimbabwe using the P32 gene[J]. Trop Anim Health Prod, 2017, 49 (1): 47- 54. doi: 10.1007/s11250-016-1156-9
9	肖雯. 绵羊痘病毒和山羊痘病毒双重PCR检测方法的建立[D]. 重庆: 西南大学, 2012.
	XIAO W. Differentiation of sheeppox virus and goatpox virus by duplex PCR[D]. Chongqing: Southwest University, 2012. (in Chinese)
10	李琛. 鉴别诊断山羊痘、绵羊痘、羊口疮和小反刍兽疫多重PCR检测方法的建立[D]. 呼和浩特: 内蒙古农业大学, 2015.
	LI C. Multiplex PCR for simultaneous detection and differentiation of goatpox, sheeppox, orf and pester des petits ruminants viruses[D]. Hohhot: Inner Mongolia Agricultural University, 2015. (in Chinese)
11	LAMIEN C E , LELENTA M , GOGER W , et al. Real time PCR method for simultaneous detection, quantitation and differentiation of capripoxviruses[J]. J Virol Methods, 2011, 171 (1): 134- 140. doi: 10.1016/j.jviromet.2010.10.014
12	YAN X M , CHU Y F , WU G H , et al. An outbreak of sheep pox associated with goat poxvirus in Gansu province of China[J]. Vet Microbiol, 2012, 156 (3/4): 425- 428.
13	TULMAN E R , AFONSO C L , ZHU L , et al. The genomes of sheeppox and goatpox viruses[J]. J Virol, 2002, 76 (12): 6054- 6061. doi: 10.1128/JVI.76.12.6054-6061.2002
14	FULZELE S , SINGH R K , HOSAMANI M , et al. Evaluation of duplex PCR and PCR-RFLP for diagnosis of sheep pox and goat pox[J]. Int J Trop Med, 2006, 1 (2): 66- 70.

引物名称Prime name		序列(5′→3′) Sequence
引物2(^#2) Primer 2	上游引物Forword primer	ACCAACACTACTTGTGCTATGA
	下游引物Reverse primer	GTATGGCATTAAGATTTGTCAACCT

引物名称 Primer		序列(5′→3′) Primer sequence	片段大小/bp Product size
引物7(PCR) Primer 7	上游引物 Forword primer	CATCATTTTGGCAAAGAATTCGCCACCATG- AATTATACGCTTAGCACAGTTAGTAGCAG	1 125
	下游引物 Reverse primer	GCTCGAGCATGCCCGGGTACCTCATTTATCGTCATCGTCTTTGT- AGTCGCTTCCTCCTCCTCCGATGCTAATACTACCAACACTACTTG	1 125

[1]	江锦秀, 张靖鹏, 林裕胜, 刘维巍, 胡奇林, 万春和. 基于Hsp70基因的绵羊肺炎支原体TaqMan检测方法的建立及其遗传演化分析[J]. 畜牧兽医学报, 2024, 55(4): 1684-1695.
[2]	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.
[3]	董欣怡, 李锦群, 陈钦玺, 廖明, 曹伟胜. J亚群禽白血病病毒血液核酸筛查技术的建立[J]. 畜牧兽医学报, 2024, 55(3): 1115-1126.
[4]	欧兰欣, 叶碧锦, 孙铭飞, 戚南山, 李娟, 吕敏娜, 林栩慧, 蔡海明, 胡俊菁, 宋勇乐, 陈祥杰, 朱易斌, 尹理君, 张健騑, 廖申权, 张浩吉. 产气荚膜梭菌四环素耐药基因双重荧光定量PCR检测方法的建立[J]. 畜牧兽医学报, 2024, 55(10): 4630-4637.
[5]	何姝凡, 邹沅彤, 黄智兰, 李倩, 降措翁西, 岳华, 汤承, 刘杰. 牛腺病毒3型感染状况及其fiber轴区基因检测[J]. 畜牧兽医学报, 2023, 54(3): 1333-1340.
[6]	邹宏, 夏应菊, 李玲, 徐璐, 赵俊杰, 王团结, 张乾义, 宋振辉. 猪瘟病毒和牛病毒性腹泻病毒双重荧光定量PCR检测方法的建立和初步应用[J]. 畜牧兽医学报, 2023, 54(10): 4422-4427.
[7]	常昊, 邱英武, 彭杰, 高琦, 宋泽布, 陈洋, 李薇, 林丽苗, 曹雪珍, 周庆丰, 张桂红, 李群辉, 郑泽中. 非洲猪瘟病毒基因Ⅰ型的双重实时荧光定量PCR检测方法建立[J]. 畜牧兽医学报, 2023, 54(10): 4428-4432.
[8]	龚三你, 蒋旭东, 马瑶, 卢建远, 字向东. 牦牛FHL2基因的克隆、组织表达与蛋白定位分析[J]. 畜牧兽医学报, 2023, 54(1): 146-156.
[9]	王华健, 张宁, 杨威, 赵志强, 李茜, 陆安, 田勇, 何欣, 赵兴华, 李杰峰. 3种食源性致病菌TaqMan多重荧光定量PCR检测方法的建立[J]. 畜牧兽医学报, 2022, 53(4): 1201-1209.
[10]	王素华, 王忠才, 黄凌哲, 莫虹斐, 吴绍强, 吕继洲, 赵治国, 帅江冰. 反刍动物埃立克体荧光定量PCR检测技术的建立和应用[J]. 畜牧兽医学报, 2021, 52(7): 1975-1982.
[11]	冯春涛, 顾文源, 王志仙, 赵增元, 朱宏波, 倪俊卿, 褚素乔, 余文莉, 李树静. 牛胚胎性别鉴定荧光定量PCR方法的优化与应用[J]. 畜牧兽医学报, 2021, 52(5): 1307-1316.
[12]	阿比克哈莫, 余忠华, 景志忠, 汤承. 检测山羊嵴病毒的实时荧光定量PCR方法的建立和应用[J]. 畜牧兽医学报, 2021, 52(5): 1349-1358.
[13]	林梦舟, 吴双, 徐建生, 谢军, 吴植, 姜勇, 朱善元. 鸭甲型肝炎病毒1型与3型双重TaqMan实时荧光定量PCR检测方法的建立与应用[J]. 畜牧兽医学报, 2021, 52(11): 3149-3156.
[14]	刘存, 吕桂霞, 徐鸿, 党安坤, 梁琳, 陈静, 孙圣福, 兰邹然. 一起境外输入性牛结节性皮肤病疫情[J]. 畜牧兽医学报, 2021, 52(11): 3317-3322.
[15]	张记宇, 韩郁茹, 时洪艳, 陈建飞, 张鑫, 刘建波, 张燎原, 冯书风, 冯廷帅, 季朝阳, 石达, 冯力. 猪急性腹泻综合征冠状病毒SYBR Green荧光定量PCR检测方法的建立及应用[J]. 畜牧兽医学报, 2021, 52(10): 2887-2894.

靶向山羊痘病毒GPCR基因SYBR Green荧光定量PCR检测方法的建立

The Establishment of a SYBR Green Fluorescence Quantitative PCR Detection Method by Targeting the G-protein Coupled Chemokine Receptor Gene of Goat Poxvirus

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

图/表 5

参考文献 14

相关文章 15

编辑推荐

Metrics

本文评价