[1]沈丽雅,潘群兴,卢凤英,等.基于P18蛋白基因的新型鸭呼肠孤病毒荧光定量RT-PCR检测方法的建立[J].江苏农业学报,2021,(06):1481-1487.[doi:doi:10.3969/j.issn.1000-4440.2021.05.016]
　SHEN Li-ya,PAN Qun-xing,LU Feng-ying,et al.Establishment of SYBR Green I fluorescence quantitative RT-PCR assay for detection of novel duck reovirus based on the gene encoding P18 protein[J].,2021,(06):1481-1487.[doi:doi:10.3969/j.issn.1000-4440.2021.05.016]
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基于P18蛋白基因的新型鸭呼肠孤病毒荧光定量RT-PCR检测方法的建立()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2021年06期
页码:
1481-1487
栏目:
畜牧兽医·水产养殖
出版日期:
2021-12-30

文章信息/Info

Title:
Establishment of SYBR Green I fluorescence quantitative RT-PCR assay for detection of novel duck reovirus based on the gene encoding P18 protein
作者:
沈丽雅1; 2; 潘群兴1; 卢凤英1; 董永毅3; 张敬峰1; 吴坤3; 刘青涛1; 姜平2; 张小飞1
（1.江苏省农业科学院兽医研究所，江苏南京210014；2.南京农业大学动物医学院，江苏南京210095；3.江苏省动物疫病预防控制中心，江苏南京210009）
Author(s):
SHEN Li-ya1; 2; PAN Qun-xing1; LU Feng-ying1; DONG Yong-yi3; ZHANG Jing-feng1; WU Kun3; LIU Qing-tao1; JIANG Ping2; ZHANG Xiao-fei1
（1.Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;2.College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China;3.Jiangsu Provincial Center for Animal Disease Control and Prevention, Nanjing 210009, China）
关键词:
新型鸭呼肠孤病毒; P18蛋白基因; 荧光定量RT-PCR
Keywords:
novel duck reovirus; gene encoding P18 protein; fluorescence quantitative RT-PCR
分类号:
S858.325.3
DOI:
doi:10.3969/j.issn.1000-4440.2021.05.016
文献标志码:
A
摘要:
根据新型鸭呼肠孤病毒（Novel duck reovirus，NDRV）特有的非结构蛋白P18基因保守序列设计1对特异性引物，建立了NDRV荧光定量RT-PCR检测方法。用该方法和普通RT-PCR方法对239份临床样品进行检测，NDRV阳性检出率分别为23.85%（57/239）和17.57%（42/239），荧光定量RT-PCR检测方法的检出率明显高于普通RT-PCR检测方法。随机取5份荧光定量RT-PCR检测方法检测的阳性的样品用鸭胚进行病毒分离培养，经普通RT-PCR扩增和基因测序证明均为NDRV。用103.0 ELD50剂量的NDRV-SY株人工感染14日龄雏鸭，不同时间采集泄殖腔肛拭子样品，通过荧光定量RT-PCR检测方法进行排毒情况监测，结果显示，雏鸭人工感染NDRV 48 h时可以从泄殖腔检测到病毒排出，持续时间可达20 d，并发现在病毒感染后的第5 d和第10 d出现2次排毒高峰。试验结果表明，建立的NDRV荧光定量RT-PCR检测方法具有良好的特异性和较高的敏感性，可用于NDRV感染的排毒监测以及临床感染的早期诊断和流行病学调查。
Abstract:
In order to rapidly detect the novel duck reovirus (NDRV), the SYBR Green I fluorescence quantitative RT-PCR method was established with a pair of specific primers designed according to the conservative gene sequence of the unique non-structural protein P18 of NDRV. 239 clinical samples were detected by the fluorescence quantitative RT-PCR method and ordinary RT-PCR methods, and the positive detection rates of the samples were 23.85% (57/239) and 17.57% (42/239), respectively. The detection rate of fluorescence quantitative RT-PCR method was significantly higher than that of ordinary RT-PCR method. Five samples were randomly selected for virus culture with duck embryo from the NDRV positive samples by the fluorescence quantitative RT-PCR method. It was proved that the five isolated viruses were NDRV by ordinary RT-PCR amplification and DNA sequencing technique. The 14-day-old ducklings were artificially infected with NDRV-SY strain at a dose of 103.0ELD50, and the cloaca swabs of these ducklings were collected for NDRV detection by this fluorescence quantitative RT-PCR at different time points. The results showed that the inoculated ducklings shed virus as early as 48 hours post inoculation, and could continue until 20 days post inoculation. In addition, two peaks of viral titers were observed at five and ten days post inoculation. Therefore, the established fluorescence quantitative RT-PCR is specific and sensitive, and can be used for the detection of NDRV, early diagnosis of clinical infection and epidemiological investigation.

参考文献/References:

[1]胡奇林，陈少莺，林锋强，等.番鸭呼肠孤病毒的鉴定[J].病毒学报，2004，20(3)：242-248.
[2]黄瑜，程龙飞，李文杨，等.雏半番鸭呼肠孤病毒的分离与鉴定[J].中国兽医学报，2004，24(1)：14-15.
[3]陈少鸾，陈仕龙，林锋强，等.一种新的鸭病(暂名鸭出血性坏死性肝炎)病原学研究初报[J].中国农学通报，2009，25(16)：28-31.
[4]刘鸿，陆新浩，任祖伊，等.番鸭“新肝病”在浙江的流行情况及防控措施[J].浙江畜牧兽医，2010，35(6)：26-28.
[5]陈少莺，陈仕龙，林锋强，等.新型鸭呼肠孤病毒的分离与鉴定[J].病毒学报，2012，28 (3)：224-230.
[6]陈宗艳，朱英奇，王世传，等.一株新型鸭源呼肠孤病毒（TH11株)的分离与鉴定[J].中国动物传染病学报，2012，20(1)：10-15.
[7]焦哲，宋彦民，孙秀秀，等.鸭疫里默氏杆菌与鸭源呼肠孤病毒混合感染的病理学诊断[J].中国兽医科学，2019，49(9)：1194- 1198.
[8]爨淑楠，徐海军，范潇会，等.一例雏番鸭大肠杆菌和呼肠孤病毒混合感染的诊治[J].中国动物保健, 2020,22(10)：44-45.
[9]陈海鹏，云涛，张存，等.新型番鸭呼肠孤病毒σC蛋白的原核表达及其抗原特性[J].浙江农业学报，2014 ,26(6):1448-1452.
[10]YUN T, HUA J G, YE W C, et al. Comparative proteomic analysis revealed complex responses to classical/novel duck reovirus infections in Cairna moschata[J]. Scientific Reports, 2018,8(1):10079.
[11]MA G, WANG D, SHI J, et al. Complete genomic sequence of reovirus isolate from Pekin ducklings in China[J].J Virol,2012,86:13137.
[12]WANG D, XU F, MA G, et al. Complete genomic sequence of a new Muscovy duck-origin reovirus from China[J].J Virol, 2012,86:12445.
[13]吴巧梅.水禽呼肠孤病毒p18蛋白鉴定及其反向遗传操作平台的建立[D].北京:中国农业科学院，2017.
[14]吴巧梅，李传峰，朱杰，等.新型鸭呼肠孤病毒P18基因的表达与鉴定[C]//中国畜牧兽医学会兽医公共卫生学分会. 中国畜牧兽医学会兽医公共卫生学分会第五次学术研讨会论文集. 通辽：中国畜牧兽医学会兽医公共卫生学分会，2016.
[15]DU X, DING M, WU Q, et al. Characterization of a P18 protein in the S1 segment of the novel duck reovirusgenome[J]. Acta Virologica, 2020,64(1):59-66.
[16]云涛，张存，华炯钢，等.鸭呼肠孤病毒基因Ⅰ型与Ⅱ型鉴别诊断RT-PCR方法的建立与应用[J].农业生物技术学报，2016，24(12):1964-1972.
[17]王锡堃，唐秀英，刘庆祥，等.应用琼扩试验检查鸡病毒性关节炎[ J]. 家畜传染病，1985(3 ):26-27.
[18]张博，陈立功，武华，等.新型鸭呼肠孤病毒 Taq Man 探针实时荧光定量 RT-PCR 检测方法的建立与应用[J]. 河北农业大学学报，2020, 43(5):80-84.
[19]晁锦，卢凤英，潘群兴，等.新型鸭呼肠孤病毒SY株的分离鉴定及培养特性[J].中国动物传染病学报，2020,28(3):1-8.

备注/Memo

备注/Memo:
收稿日期：2021-03-19基金项目：国家重点研发计划项目（2017YFD0500804）；江苏现代农业（水禽）产业技术体系疾病防控创新团队项目[JATS(2020)321]作者简介：沈丽雅（1996-），女，浙江湖州人，硕士研究生，主要从事动物传染病防治研究。（Tel）15205172927；（E-mail）823320658@qq.com通讯作者：张小飞，（E-mail）xiaofei0804@sina.com；姜平，(E-mail) jiangp@ njau.edu.cn
更新日期/Last Update: 2022-01-07