[1]吴双,姜勇,徐建生,等.鸭坦布苏病毒、鸭肠炎病毒和番鸭细小病毒TaqMan三重实时荧光定量PCR检测方法的建立与临床应用[J].江苏农业学报,2020,(03):626-633.[doi:doi:10.3969/j.issn.1000-4440.2020.03.014]
　WU Shuang,JIANG Yong,XU Jian-sheng,et al.Establishment and clinical application of triple TaqMan real-time fluorescence quantitative PCR detection method for duck Tembusu virus, duck enteritis virus and Muscovy duck parvovirus[J].,2020,(03):626-633.[doi:doi:10.3969/j.issn.1000-4440.2020.03.014]
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鸭坦布苏病毒、鸭肠炎病毒和番鸭细小病毒TaqMan三重实时荧光定量PCR检测方法的建立与临床应用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2020年03期
页码:
626-633
栏目:
畜牧兽医·水产养殖
出版日期:
2020-06-30

文章信息/Info

Title:
Establishment and clinical application of triple TaqMan real-time fluorescence quantitative PCR detection method for duck Tembusu virus, duck enteritis virus and Muscovy duck parvovirus
作者:
吴双1; 姜勇1; 2; 徐建生2; 吴植1; 谢军1; 宋海港1; 2; 魏若瑶2; 高悦1; 朱善元1
（1.江苏农牧科技职业学院江苏省兽用生物制药高技术研究重点实验室，江苏泰州225300；2.扬州大学兽医学院，江苏扬州225009）
Author(s):
WU Shuang1; JIANG Yong1; 2; XU Jian-sheng2; WU Zhi1; XIE Jun1; SONG Hai-gang1; 2; WEI Ruo-yao2; GAO Yue1; ZHU Shan-yuan1
（1.Jiangsu Agri-animal Husbandry Vocational College/Jiangsu Provincial Key Laboratory of Veterinary Bio-pharmaceutical High-tech Research，Taizhou 225300， China;2.College of Veterinary Medicine, Yangzhou University, Yangzhou 225009， China）
关键词:
鸭坦布苏病毒; 鸭肠炎病毒; 番鸭细小病毒; TaqMan实时荧光定量PCR
Keywords:
duck Tembusu virus; duck enteritis virus; Muscovy duck parvovirus; TaqMan real-time fluorescence quantitative PCR
分类号:
S834
DOI:
doi:10.3969/j.issn.1000-4440.2020.03.014
文献标志码:
A
摘要:
本研究旨在建立一种快速、敏感和高特异性检测鸭坦布苏病毒（DTMUV）、鸭肠炎病毒（DEV）和番鸭细小病毒（MDPV）的TaqMan三重实时荧光定量PCR（q-PCR）的诊断方法并应用于临床疑似样品检测。根据DTMUV的E基因、DEV的UL2基因和MDPV的VP3基因保守区域，分别设计合成了3对特异性引物和探针，在建立单重q-PCR方法的基础上建立了三重q-PCR方法。运用三重q-PCR方法对198份来自江苏和安徽的鸭组织疑似病料进行检测，结果表明，建立的TaqMan三重q-PCR可同时检测这3种病毒，检测灵敏度至少达100个拷贝，相关系数（R2）均在0.99以上，扩增效率为90%～110%。同时，该方法对H9亚型禽流感病毒（H9N2 AIV）、鸭甲肝病毒Ⅰ型（DHAV-1）、番鸭呼肠孤病毒（MDRV）、鸭呼肠孤病毒（DRV）、新城疫病毒（NDV）、鹅细小病毒（GPV）的检测均为阴性，表明该方法具备特异性强、灵敏度高、重复性好和快速等优点。与常规PCR检测方法相比，三重q-PCR方法灵敏度大约高100倍。临床疑似样品检测结果表明DTMUV的检出率最高，3种病毒混合感染亦常见。建立的TaqMan三重q-PCR检测方法为DTMUV、DEV和MDPV的临床样品检测提供了快速、有效、特异和灵敏的工具，也为临床分子流行病学调查及定量分析奠定了基础。
Abstract:
This study aimed to establish a triple TaqMan real-time fluorescence quantitative PCR(q-PCR) method for the rapid, sensitive and specific detection of duck Tembusu virus (DTMUV), duck enteritis virus (DEV) and Muscovy duck parvovirus (MDPV). Based on the conserved regions of DTMUV E, DEV UL2 and MDPV VP3 genes, three pairs of specific primers and probes were designed and synthesized respectively. Furthermore, a triple q-PCR method was established based on the establishment of a single q-PCR method. The triple q-PCR method was used to detect 198 suspected samples from Jiangsu and Anhui. The results showed that the established TaqMan triple q-PCR method could detect these three viruses simultaneously. The detection sensitivity was at least 100 copies, the correlation coefficient (R2) was above 0.99, and the amplification efficiency ragned from 90% to 110%. At the same time, the detection results of H9N2 avian influenza virus (H9N2 AIV), duck hepatitis A virus type I (DHAV-1), Muscovy duck reovirus (MDRV), duck reovirus (DRV), Newcastle disease virus (NDV) and goose parvovirus (GPV) were negative, it showed that the method had the advantages of high specificity, high sensitivity fastness good repeatability. The sensitivity of triple q-PCR method was approximately 100 times greater than that of conventional PCR assay. The results of clinical suspected samples indicated that the detection rate DTMUV was highest, and mixed infections of three viruses were also common. This triple TaqMan q-PCR assay provides a fast, efficient, specific and sensitive tool for the detection of DTMUV, DEV, MDPV and also lays the foundation for clinical molecular epidemiological investigation and quantitative analysis.

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备注/Memo

备注/Memo:
收稿日期：2019-10-18基金项目：江苏省“333工程”项目（BRA2018376）；江苏省“333”高层次人才培养工程项目；江苏省农业科技自主创新基金项目[CX（18）1004]；江苏省“六大人才高峰”项目（NY-009）；大学生创新创业训练计划项目(201912806030Y)作者简介：吴双(1983-), 女, 江苏徐州人, 博士, 教授, 主要从事动物传染病防控和兽用基因工程疫苗研究。（Tel）0523-86356818；（E-mail）jswushuang@sina.cn。姜勇为共同第一
更新日期/Last Update: 2020-07-14