检测山羊嵴病毒的实时荧光定量PCR方法的建立和应用

doi:10.11843/j.issn.0366-6964.2021.05.020

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (5): 1349-1358.doi: 10.11843/j.issn.0366-6964.2021.05.020

检测山羊嵴病毒的实时荧光定量PCR方法的建立和应用

阿比克哈莫¹, 余忠华², 景志忠^1*, 汤承^3*

1. 中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室农业部兽医公共卫生重点实验室, 兰州 730046;
2. 阿坝藏族羌族自治州动物科学技术研究所, 红原 624400;
3. 西南民族大学畜牧兽医学院, 成都 610041

收稿日期:2020-11-03 出版日期:2021-05-23 发布日期:2021-05-22
通讯作者: 景志忠,主要从事人兽共患病以及动物疫苗与分子免疫学研究,Tel:0931-8341979,E-mail:jingzhizhong@caas.cn;汤承,主要从事动物病原生物学研究,Tel:028-85528276,E-mail:tangcheng101@163.com
作者简介:阿比克哈莫(1991-),女,四川凉山人,博士生,主要从事动物疫苗和分子免疫学研究,E-mail:274577380@qq.com
基金资助:
“十三五”国家重点研发计划（2016YFD0500907）；西南民族大学新发动物疫病创新团队（2020 NTD02）；动物医学四川省高等学校重点实验室平台项目（2020PTJS29001）

Establishment and Application of a Real Time Fluorescent Quantitative PCR Assay for Detecting Caprine Kobuvirus

ABI-Kehamo¹, YU Zhonghua², JING Zhizhong^1*, TANG Cheng^3*

1. State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Public Health of Ministry of Agriculture and Rural Affairs, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China;
2. Institute of Animal Science and Technology of Aba Tibetan and Qiang Autonomous Prefecture, Hongyuan 624400, China;
3. College of Animal Science and Veterinary Medicine, Southwest Minzu University, Chengdu 610041, China

Received:2020-11-03 Online:2021-05-23 Published:2021-05-22

摘要/Abstract

摘要： 山羊嵴病毒（caprine kobuvirus，CKoV）是国内山羊新发病毒，本试验目的是建立检测CKoV的实时荧光定量PCR方法，并对西南三省腹泻山羊粪样进行该病毒的检测。选择CKoV最保守的3D基因序列，设计3D基因引物，初步设计实时荧光定量PCR，经优化反应条件和反应体系，成功建立了检测CKoV的TB Green染料法实时荧光定量PCR方法。该方法在病毒浓度2.23×10²～2.23×10⁸copies·μL^-1范围内线性关系良好，相关系数为0.999 2，扩增效率为110%；此方法具有良好的特异性和稳定性，最低检测限为2.23×10¹copies·μL^-1。采用所建立的方法对2020年1—4月采集自四川、云南和重庆的共15个场79份山羊腹泻粪样本进行检测，结果CKoV的平均检出率为25.3%，场阳性率为53.3%。并且本试验获得了13个完整的CKoV 3D基因，遗传进化分析表明这13个3D基因具有独特的进化趋势。本研究为CKoV的分子检测提供了一种新的技术手段和基础流行病学数据。

关键词: 山羊嵴病毒, 实时荧光定量PCR方法, 山羊, 腹泻

Abstract: Caprine kobuvirus (CKoV) is an emerging virus in goats in China, this study aimed to establish a real-time fluorescent quantitative PCR assay for detecting CKoV. Through designing primers targeting the 3D gene of CKoV and optimizing the reaction conditions and system, a TB Green Fluorescent Quantitative PCR assay was successfully established. The assay had a good linear relationship with CKoV in the range of 2.23×10²-2.23×10⁸copies·μL^-1 with a correlation coefficient (R²) of 0. 999 2, and its amplification efficiency was 110%. Moreover, the assay has good specificity and stability, and the detection limit was 2.23×10¹copies·μL^-1. Furthermore, out of 79 diarrhea samples of lambs collected from Sichuan, Chongqing, and Yunnan regions from January 2020 to April 2020, 25.3%samples (20/79) were detected as CKoV positive, and the farms’ positive rate was 53.3% (8/15). In addition, 13 complete 3D genes of CKoV were obtained in this study, and evolution analysis showed that these 13 3D genes had unique evolutionary trends. In conclusion, this study provides a new molecular method for detecting CKoV and basic epidemiological data of domestic CKoV.

Key words: caprine kobuvirus, real time fluorescent quantitative PCR assay, goat, diarrhea

中图分类号:

S852.659.6

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.

ABI-Kehamo, YU Zhonghua, JING Zhizhong, TANG Cheng. Establishment and Application of a Real Time Fluorescent Quantitative PCR Assay for Detecting Caprine Kobuvirus[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(5): 1349-1358.

参考文献 28

[1]	KANG S, KIM Y C. The genome-based virus taxonomy with ICTV database extension[J]. Genomics Inform, 2018, 16(4):e22.
[2]	YANG F, LIU X W, ZHOU Y C, et al. Histopathology of Porcine kobuvirus in Chinese piglets[J]. Virol Sin, 2015, 30(5):396-399.
[3]	ZHU L, WANG X X, REN J S, et al. Structure of human Aichi virus and implications for receptor binding[J]. Nat Microbiol, 2016, 1(11):16150.
[4]	WANG L Y, FREDRICKSON R, DUNCAN M, et al. Bovine kobuvirus in calves with diarrhea, United States[J]. Emerg Infect Dis, 2020, 26(1):176-178.
[5]	CHEN Y S, CHEN B C, LIN Y S, et al. Detection of Aichi virus with antibody targeting of conserved viral protein 1 epitope[J]. Appl Microbiol Biotechnol, 2013, 97(19):8529-8536.
[6]	ZHAI S L, ZHANG H, LIN T, et al. A novel porcine kobuvirus emerged in piglets with severe diarrhoea in China[J]. Transbound Emerg Dis, 2017, 64(4):1030-1036.
[7]	BARRY A F, RIBEIRO J, ALFIERI A F, et al. First detection of kobuvirus in farm animals in Brazil and the Netherlands[J]. Infect Genet Evol, 2011, 11(7):1811-1814.
[8]	CHAROENKUL K, JANETANAKIT T, CHAIYAWONG S, et al. First detection and genetic characterization of canine kobuvirus in domestic dogs in Thailand[J]. BMC Vet Res, 2019, 15(1):254.
[9]	REUTER G, KECSKEMÉTI S, PANKOVICS P. Evolution of porcine kobuvirus infection, Hungary[J]. Emerging Infect Dis, 2010, 16(4):696-698.
[10]	COLLINS P J, MCMENAMY M J, MCCLINTOCK J, et al. Molecular detection of kobuviruses in livestock in Northern Ireland and the Republic of Ireland[J]. Arch Virol, 2017, 162(5):1275-1279.
[11]	LEE M H, JEOUNG H Y, LIM J A, et al. Kobuvirus in South Korean black goats[J]. Virus Genes, 2012, 45(1):186-189.
[12]	OEM J K, LEE M H, LEE K K, et al. Novel Kobuvirus species identified from black goat with diarrhea[J]. Vet Microbiol, 2014, 172(3-4):563-567.
[13]	MELEGARI I, DI PROFIO F, SARCHESE V, et al. First molecular evidence of kobuviruses in goats in Italy[J]. Arch Virol, 2016, 161(11):3245-3248.
[14]	ABI K M, ZHANG Q, JING Z Z, et al. First detection and molecular characteristics of caprine kobuvirus in goats in China[J]. Infect Genet Evol, 2020, 85:104566.
[15]	DI MARTINO B, DI PROFIO F, MELEGARI I, et al. Molecular detection of kobuviruses in European roe deer (Capreolus capreolus) in Italy[J]. Arch Viroly, 2015, 160(8):2083-2086.
[16]	AMBERT-BALAY K, LORROT M, BON F, et al. Prevalence and genetic diversity of Aichi virus strains in stool samples from community and hospitalized patients[J]. J Clin Microbiol, 2008, 46(4):1252-1258.
[17]	LODDER W J, RUTJES S A, TAKUMI K, et al. Aichi virus in sewage and surface water, the Netherlands[J]. Emerging Infect Dis, 2013, 19(8):1222-1230.
[18]	孟丽, 陶洁, 李本强, 等. 猪7种腹泻相关病毒的临床检测及猪嵴病毒的遗传进化分析[J]. 生物工程学报, 2017, 33(8):1292-1303.MENG L, TAO J, LI B Q, et al. Clinical detection of seven porcine diarrhea-associated viruses and evolution analysis of porcine kobuvirus[J]. Chinese Journal of Biotechnology, 2017, 33(8):1292-1303. (in Chinese)
[19]	胡军勇, 汤细彪, 胡睿铭, 等. 猪库布病毒RT-PCR检测方法的建立及湖北省流行病学初步调查[J]. 华中农业大学学报, 2012, 31(4):485-489.HU J Y, TANG X B, HU R M, et al. Porcine kobuvirus RT-PCR detection assay establishment and application in primary epidemiology investigation in Hubei Province[J]. Journal of Huazhong Agricultural University, 2012, 31(4):485-489. (in Chinese)
[20]	李慧平, 汤承, 岳华. 实时荧光定量RT-PCR检测牛库布病毒[J]. 畜牧兽医学报, 2020, 51(4):888-893.LI H P, TANG C, YUE H. Development and application of a real-time RT-PCR assay for detecting bovine kobuvirus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(4):888-893. (in Chinese)
[21]	REUTER G, BOLDIZSÁR Á, PANKOVICS P. Complete nucleotide and amino acid sequences and genetic organization of porcine kobuvirus, a member of a new species in the genus Kobuvirus, family Picornaviridae[J]. Arch Virol, 2009, 154(1):101-108.
[22]	LIU P J, LI P, LYU W T, et al. Epidemiological study and variation analysis of the porcine kobuvirus 3D gene in Sichuan Province, China[J]. Virol Sin, 2015, 30(6):460-463.
[23]	郭紫晶, 何琪富, 汤承, 等. 检测纽布病毒的Real-time RT-PCR方法的建立与应用[J]. 畜牧兽医学报, 2019, 50(4): 893-900.GUO Z J, HE Q F, TANG C, et al. Establishment and application of a real-time RT-PCR assay for detecting bovine Nebovirus[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(4): 893-900. (in Chinese)
[24]	NIU T J, YI S S, WANG X, et al. Detection and genetic characterization of kobuvirus in cats: the first molecular evidence from Northeast China[J]. Infect Genet Evol, 2019, 68:58-67.
[25]	NANTEL-FORTIER N, LACHAPELLE V, LETELLIER A, et al. Kobuvirus shedding dynamics in a swine production system and their association with diarrhea[J]. Vet Microbiol, 2019, 235:319-326.
[26]	谢荣辉, 刘霞, 赵灵燕, 等. 猪嵴病毒荧光定量RT-PCR检测方法的建立和应用[J]. 中国兽医科学, 2018, 48(4):438-442.XIE R H, LIU X, ZHAO L Y, et al. Establishment and application of the real-time reverse transcription polymerase chain reaction for detection of porcine kobuvirus[J]. Chinese Veterinary Science, 2018, 48(4):438-442. (in Chinese)
[27]	PARK S J, KIM H K, SONG D S, et al. Molecular detection and genetic characterization of kobuviruses in fecal samples collected from diarrheic cattle in Korea[J]. Infect Genet Evol, 2011, 11(5):1178-1182.
[28]	DUBANKOVA A, HOROVA V, KLIMA M, et al. Structures of kobuviral and siciniviral polymerases reveal conserved mechanism of picornaviral polymerase activation[J]. J Struct Biol, 2019, 208(2):92-98.

检测山羊嵴病毒的实时荧光定量PCR方法的建立和应用

Establishment and Application of a Real Time Fluorescent Quantitative PCR Assay for Detecting Caprine Kobuvirus

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献 28

相关文章 15

编辑推荐

Metrics

本文评价

[1]	严晓春, 习海娇, 李金泉, 王志英, 苏蕊. 内蒙古绒山羊绒毛性状基因组育种值估计准确性研究[J]. 畜牧兽医学报, 2024, 55(1): 120-128.
[2]	张晨俭, 李隐侠, 丁强, 刘伟佳, 王慧利, 何南, 吴家顺, 曹少先. CRISPR/Cas9技术高效制备山羊SOCS2基因编辑胚胎[J]. 畜牧兽医学报, 2024, 55(1): 129-141.
[3]	张德安, 杨若渚, 刘杰, 刘德武, 邓铭, 柳广斌, 孙宝丽, 郭勇庆, 李耀坤. 饲喂青贮黄梁木代替青贮玉米川中黑山羊肝转录组的表达分析[J]. 畜牧兽医学报, 2024, 55(1): 232-244.
[4]	周建浩, 王东方, 刘影, 王淑娟, 马震原, 谢彩华, 赵雪丽, 杨海波, 冯桂丹, 康台生, 胡煜锋, 李博文, 闫若潜. 猪流行性腹泻病毒微滴式数字PCR定量检测方法的建立及初步应用[J]. 畜牧兽医学报, 2024, 55(1): 413-418.
[5]	唐崟梅, 李琪, 李海洋, 林亚秋, 王永, 向华, 黄炼, 朱江江. 山羊FATP2基因的克隆及对前体脂肪细胞脂质沉积的影响[J]. 畜牧兽医学报, 2023, 54(9): 3642-3652.
[6]	邵鹏, 唐崟梅, 林亚秋, 王永, 向华, 黄炼, 朱江江. PSMD9对山羊前体脂肪细胞脂质沉积的调控作用研究[J]. 畜牧兽医学报, 2023, 54(9): 3653-3663.
[7]	姜玲玲, 牛小霞, 刘强, 张刚, 王璞, 李勇. 宁夏地区肉牛腹泻相关病毒感染状况的分析[J]. 畜牧兽医学报, 2023, 54(9): 3863-3871.
[8]	刘华, 殷冬冬, 邵颖, 宋祥军, 王振宇, 潘孝成, 涂健, 何长生, 朱良强, 祁克宗. 猪流行性腹泻病毒RAA-CRISPR/Cas13a检测方法的建立与初步应用[J]. 畜牧兽医学报, 2023, 54(9): 3991-3997.
[9]	王思盈, 邹宏, 宋振辉. Na⁺/H⁺交换体家族第三个亚型在感染性腹泻中的作用及活性调控机制[J]. 畜牧兽医学报, 2023, 54(8): 3230-3241.
[10]	黄江, 李闯, 崔月琦, 袁雪莹, 赵志诚, 刘宇, 周玉龙, 朱战波, 张泽财. 基于小鼠模型研究肠道菌群紊乱对BVDV易感性的影响[J]. 畜牧兽医学报, 2023, 54(8): 3466-3473.
[11]	张任豹, 周东辉, 周李生, 高霄霄, 柳楠, 贺建宁. 基于70 K SNP芯片分析济宁青山羊保种群体的遗传结构[J]. 畜牧兽医学报, 2023, 54(7): 2836-2847.
[12]	周炜炜, 王雪峰, 张梦洁, 杨娟, 孙悦龙, 张祖烽, 张宇欣, 豆佳红, 王梓颖, 戴小枫, 李秀梅. 基于生物网络功能模块整合组方规律分析四黄止痢颗粒治疗仔猪腹泻的作用机制[J]. 畜牧兽医学报, 2023, 54(7): 3031-3043.
[13]	邵钺馨, 张新钰, 葛丽岩, 史怀平. 西农萨能奶山羊ATF4基因克隆及其功能初步研究[J]. 畜牧兽医学报, 2023, 54(6): 2353-2364.
[14]	刘杰, 许香萍, 邓铭, 邹娴, 江声伟, 刘德武, 柳广斌, 孙宝丽, 郭勇庆, 李耀坤. miR-144-5p靶向WNT5a对山羊卵巢颗粒细胞增殖、凋亡的影响[J]. 畜牧兽医学报, 2023, 54(6): 2421-2435.
[15]	张家祺, 甲呷格西, 周群, 宋鑫, 张斌. 宏基因组学技术分析四川省集约化猪场中仔猪腹泻粪便的病毒种群[J]. 畜牧兽医学报, 2023, 54(6): 2478-2486.