[1]杨晓宇,陈世界,林华,等.多重TaqMan荧光定量PCR检测仔猪先天性震颤相关病毒方法的建立与应用[J].江苏农业学报,2020,(04):971-977.[doi:doi:10.3969/j.issn.1000-4440.2020.04.023]
　YANG Xiao-yu,CHEN Shi-jie,LIN Hua,et al.Establishment and application of multiple TaqMan fluorescence quantitative PCR method for detection of congenital tremor related viruses in piglets[J].,2020,(04):971-977.[doi:doi:10.3969/j.issn.1000-4440.2020.04.023]
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多重TaqMan荧光定量PCR检测仔猪先天性震颤相关病毒方法的建立与应用()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2020年04期
页码:
971-977
栏目:
畜牧兽医·水产养殖
出版日期:
2020-08-31

文章信息/Info

Title:
Establishment and application of multiple TaqMan fluorescence quantitative PCR method for detection of congenital tremor related viruses in piglets
作者:
杨晓宇1; 陈世界2; 林华2; 张婧2; 安微2; 朱玲1; 3
(1.四川农业大学动物医学院，四川成都611130；2.成都海关检验检疫技术中心，四川成都610041；3.四川农业大学动物疫病与人类健康四川省重点实验室，四川成都611130）
Author(s):
YANG Xiao-yu1; CHEN Shi-jie2; LIN Hua2; ZHANG Jing2; AN Wei2; ZHU Ling1; 3
(1.College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China;2.Chengdu Customs Inspection and Quarantine Technology Center, Chengdu 610041, China;3.Sichuan Key Laboratory of Animal Diseases and Human Health, Sichuan Agricultural University, Chengdu 611130, China)
关键词:
猪先天性震颤; 猪非典型瘟病毒; 猪瘟病毒; 猪圆环病毒3型; 猪捷申病毒1型; 多重TaqMan荧光定量PCR检测
Keywords:
congenital tremor; atypical porcine pestivirus; classical swine fever virus; porcine circoviruses type 3; porcine teschovirus 1; multiple TaqMan fluorescence quantitative PCR
分类号:
S852.65+1
DOI:
doi:10.3969/j.issn.1000-4440.2020.04.023
文献标志码:
A
摘要:
旨在研究能够快速、准确、灵敏地鉴别、诊断引起仔猪先天性震颤的猪非典型瘟病毒(Atypical porcine pestivirus, APPV）、猪瘟病毒(Classical swine fever virus，CSFV）、猪圆环病毒3型(Porcine circoviruses type 3, PCV-3）及猪捷申病毒1型(Porcine teschovirus 1，PTV-1）的方法。通过对GenBank中登录的APPV的NS3基因序列、CSFV的E2基因序列、PCV-3的ORF2基因序列和PTV-1的VP1基因序列进行分析，设计针对4种病毒的特异性引物和TaqMan水解探针。通过对反应条件和反应程序进行优化，拟建立针对仔猪先天性震颤相关病毒的多重TaqMan荧光定量PCR检测方法。结果显示，研究得到的多重TaqMan荧光定量PCR检测方法能够特异性检测APPV、CSFV、PCV-3和PTV-1，而与其他病原无交叉反应；该研究方法对APPV、CSFV、PCV-3和PTV-1的最低检测量分别为1 μl 9.2×102拷贝、48拷贝、56拷贝和17拷贝。重复性试验结果显示，组内、组间变异系数均小于2.10%，重复性和稳定性较为突出。用该方法对273份临床样品进行检测，结果显示，APPV、CSFV、PTV-1和PCV-3的阳性率分别为20.5%、2.9%、1.5%和10.6%，其中APPV、CSFV二者共同感染的检出率为1.5%，APPV、PCV-3二者共同感染的检出率为4.4%，APPV、PTV-1二者共同感染的检出率为1.5%，APPV、CSFV、PCV-3共同感染的检出率为1.1%。该检测方法操作简单、耗时短、不易对环境造成污染，检测结果灵敏、准确，基于该方法对四川地区仔猪先天性震颤相关病毒共感染情况进行的流行病学调查，可以为后期研究提供重要的数据基础。
Abstract:
The aim of this study was to develop a rapid, accurate and sensitive method for the identification and diagnosis of atypical porcine pestivirus (APPV), classical swine fever virus (CSFV), porcine circovirus type 3 (PCV-3) and porcine teschovirus 1 (PTV-1) that caused congenital tremor in piglets. By analyzing the NS3 gene sequence of APPV, E2 gene sequence of CSFV, ORF2 gene sequence of PCV-3 and VP1 gene sequence of PTV-1 registered in GenBank, specific primers and TaqMan hydrolysis probes for four viruses were designed. Multiple TaqMan fluorescence quantitative PCR detection method for congenital tremor related viruses in piglets was established by optimizing the reaction conditions and procedures. The results showed that APPV, CSFV, PCV-3 and PTV-1 could be specifically detected by the obtained multiple TaqMan fluorescence quantitative PCR detection method, without cross-reaction with other pathogens. By using the research method, the minimum detectable amount of APPV, CSFV, PCV-3 and PTV-1 was 9.2×102copies per microlitre, 48.0 copies per microlitre, 56.0 copies per microlitre and 17.0 copies per microlitre, respectively. Results of the repeatability test showed that the coefficients of variation between and within groups were less than 2.10%, and the repeatability and stability were outstanding. The method was used to detect APPV, CSFV, PCV-3 and PTV-1 in 273 clinical samples, the results indicated that the positive rates of APPV, CSFV, PTV-1 and PCV-3 were 20.5%, 2.9%, 1.5% and 10.6%, respectively. In addition, the detection rate of APPV and CSFV co-infection was 1.5%, the detection rate of APPV and PCV-3 co-infection was 4.4%, the detection rate of APPV and PTV-1 co-infection was 1.5%, and the detection rate of APPV, CSFV and PCV-3 joint-infection was 1.1%. The detection method is simple, time-saving and difficult to pollute the environment, and the detection results are sensitive and accurate. An epidemiological investigation of congenital tremor-associated virus infection in piglets of Sichuan area based on this method can provide important data basis for later research.

参考文献/References:

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备注/Memo

备注/Memo:
收稿日期：2020-01-13基金项目：“十三五”国家重点研发计划项目(2018YFD0501102）；四川省科技支撑计划项目(2018NZ0150）；四川省“十三五”育种攻关计划项目(2016NYZ0052）；国家农业产业技术体系四川兽药创新团队专项(CARS-SVDIP）作者简介：杨晓宇(1993-），男，山东烟台人，硕士研究生，主要从事动物传染病病原分子生物学研究。(E-mail）1491618529@qq.com
更新日期/Last Update: 2020-09-08