[1]白彩霞,张达,赵靓,等.鹅星状病毒SYBR Green I荧光定量RT-PCR方法的建立[J].江苏农业学报,2020,(03):634-638.[doi:doi:10.3969/j.issn.1000-4440.2020.03.015]
　BAI Cai-xia,ZHANG Da,ZHAO Liang,et al.Development and application of SYBR Green I fluorescence quantitative RT-PCR assay for detection of goose astrovirus[J].,2020,(03):634-638.[doi:doi:10.3969/j.issn.1000-4440.2020.03.015]
点击复制

鹅星状病毒SYBR Green I荧光定量RT-PCR方法的建立()
分享到：

江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2020年03期
页码:
634-638
栏目:
畜牧兽医·水产养殖
出版日期:
2020-06-30

文章信息/Info

Title:
Development and application of SYBR Green I fluorescence quantitative RT-PCR assay for detection of goose astrovirus
作者:
白彩霞1; 张达1; 赵靓1; 杨侃侃1; 王小朋1; 李新路1; 李锦春1; 李永东2; 王勇1
（1.安徽农业大学动物科技学院,安徽合肥230036；2.宁波市疾病预防控制中心，浙江宁波315010)
Author(s):
BAI Cai-xia1; ZHANG Da1; ZHAO Liang1; YANG Kan-kan1; WANG Xiao-peng11; LI Xin-lu1; LI Jin-chun1; LI Yong-dong2; WANG Yong1
(1.College of Animal Science and Technology, Anhui Agricultural University, Hefei 230036, China;2.Ningbo Municipal Center for Disease Control and Prevention, Ningbo 315010, China)
关键词:
鹅星状病毒; SYBR Green I 荧光定量RT-PCR; 检测
Keywords:
goose astrovirus(GAstV); SYBR Green I flucrescence quantitative RT-PCR; detection
分类号:
S858.33
DOI:
doi:10.3969/j.issn.1000-4440.2020.03.015
文献标志码:
A
摘要:
为建立快速检测鹅星状病毒(GAstV)的方法，本研究根据GenBank中GAstV ORF2基因序列设计1对特异性引物，构建重组质粒pMD19-T-GAstV，以其作为标准品建立了GAstV的SYBR Green I荧光定量RT-PCR检测方法。优化其反应条件及体系，进行特异性、敏感性和重复性试验以及临床样本检测。试验结果显示，除GAstV外，禽白血病病毒(ALV)、血清4型禽腺病毒(FAdV-4)、鸡传染性喉气管炎病毒(ILTV)和鸡传染性贫血病病毒(CIAV)等常见禽病病原利用该方法检测均呈阴性，表明其特异性良好;建立的荧光定量RT-PCR检出的最低模板含量为1 μl 3.75×101拷贝，敏感性较高;组内和组间重复性试验变异系数均小于1 %。利用该方法对来自安徽地区的32份临床样品进行检测，阳性检出率为43.75%，常规PCR方法阳性检出率为18.75%，阳性检出符合率100%，表明该方法可用于临床样品检测。该方法的建立为临床样品中GAstV的快速高效检测提供了技术支持。
Abstract:
In order to rapidly detect the goose astrovirus (GAstV), the SYBR Green I fluorescence quantitative RT-PCR method was established. Firstly, a pair of specific primers was designed based on the ORF2 gene sequence of the GAstV in GenBank. Secondly, the recombinant plasmid pMD19-T-GAstV was constructed and used as a standard template to generate the standard curve. The reaction conditions and reaction system were optimized, the specificity, sensitivity and reproducibility of the assay were tested. Moreover, the established assay was used in clinical samples detection. The results showed that the detection results of fowl adenovirus serotype 4(FAdV-4), infectious laryngotracheitis virus(ILTV), avian leukosis virus(ALV), chicken infectious anemia virus(CIAV) and other common avian pathogens were negative except the GAstV. It indicated that the assay had good specificity. The lowest template content detected by fluorescence quantitative RT-PCR assay was 3.75×101 copies per milliliter. The variation coefficients in the repeatability test were less than 1%. Thirty-two clinical samples from Anhui province were tested using this assay. The positive detection rate was 43.75%, the positive rate of conventional PCR was 18.75%, and the coincidence rate was 100%. In conclusion, the established assay provides technical support for the rapid and efficient detection of GAstV in clinical samples.

参考文献/References:

[1]YUAN X Y, MENG K, ZHANG Y X, et al. Genome analysis of newly emerging goose-origin nephrotic astrovirus in China reveals it belongs to a novel genetically distinct astrovirus[J]. Infect Genet Evol, 2019, 67:1-6.
[2]PANTINJACKWOOD M J, STROTHER K O, MUNDT E, et al. Molecular characterization of avian astroviruses[J]. Arch Virol, 2011, 156(2):235-244.
[3]SUN N, YANG Y, WANG G S, et al. Detection and characterization of avastrovirus associated with diarrhea isolated from minks in China[J]. Food Environ Virol, 2014, 6(3): 169-174.
[4]MARINA B, DARKO M, ZDENKO B, et al. Genetic characterization of turkey astroviruses identified in Croatian poultry flocks[J]. Vet Archiv, 2013,83(1):57-67.
[5]MARY J P, ERICA S, PETER R W. Molecular characterization and typing of chicken and turkey astroviruses circulating in the United States: Implications for diagnostics.[J]. Avian Dis, 2006, 50(3):397-404.
[6]NIU X Y, TIAN J J, YANG J, et al. Novel goose astrovirus associated gout in gosling, China.[J]. Vet Microbiol, 2018, 220:53-56.
[7]ZHANG Q S, CAO Y X, WANG J, et al. Isolation and characterization of an astrovirus causing fatal visceral gout in domestic goslings[J]. Emerg Microbes Infect, 2018, 7(1):71.
[8]ZHANG X Y, REN D, LI T F, et al. An emerging novel goose astrovirus associated with gosling gout disease, China[J]. Emerg Microbes Infect, 2018, 7(1):152.
[9]YUAN X Y, MENG K, ZHANG Y X, et al. Establishment and application of rapid diagnosis for reverse transcription-quantitative PCR of newly emerging goose-origin nephrotic astrovirus in China[J]. mSphere, 2018, 3(6):e00380.
[10]IAN M M, KATHERINE E A, ANDREAS N. Real-time PCR in virology[J]. Nucleic Acids Res, 2002, 30(6): 1292-1305.
[11]周红英. 一例雏鹅痛风病的诊治[J]. 畜禽业， 2017(7):120-121.
[12]陈英辉. 鹅痛风的发生原因与诊治[J]. 畜牧兽医科技信息, 2017(4):112.
[13]杨慧赞，童桂香，郑晓聪，等. IHHNV PCR检测方法改进及广西流行株基因型分析[J].南方农业学报，2019,50(5):1127-1132.
[14]楚会萌，任亚初，程凯慧，等. 一株牛流行热病毒的分离与鉴定[J].山东农业科学，2019，51(3):111-114.
[15]彭昊，吴翠兰，李军，等. 广西黄牛乳头状瘤病毒的鉴定及其基因组序列分析[J]. 南方农业学报，2018,49(3): 563-571.
[16]周晓俊,朱淮民,邱璐. 检测蚊虫感染黄病毒属病毒Real-time PCR方法的建立[J]. 中国病原生物学杂志, 2008, 3(8):561-563.
[17]CHANDRA B P, RISHI S, VIJAI K G, et al. Q-PCR based culture-independent enumeration and detection of enterobacter: an emerging environmental human pathogen in riverine systems and potable water[J]. Front Microbiol, 2016, 7:172.
[18]DAVID N, MARIO A, LLOREN G R, et al. Dynamics of Torque teno sus virus 1 (TTSuV1) and 2 (TTSuV2) DNA loads in serum of healthy and postweaning multisystemic wasting syndrome (PMWS) affected pigs[J]. Vet Microbiol, 2011, 152(3/4):284-290.

相似文献/References:

[1]吕炫,张淼,胡增金,等.鹅星状病毒衣壳纤突蛋白VP27多克隆抗体的制备和鉴定[J].江苏农业学报,2021,(02):412.[doi:doi:10.3969/j.issn.1000-4440.2021.02.018]
　LYU Xuan,ZHANG Miao,HU Zeng-jin,et al.Preparation and identification of polyclonal antibody against the capsid spike protein VP27 of goose astrovirus[J].,2021,(03):412.[doi:doi:10.3969/j.issn.1000-4440.2021.02.018]

备注/Memo

备注/Memo:
收稿日期：2019-08-26基金项目：国家自然科学基金项目（31602063）作者简介：白彩霞（1995-），女，土族，青海西宁人，硕士研究生，主要从事动物传染病研究。（E-mail）baicaixiayx@126.com。张达为共同第一作者。通讯作者：李永东，(E-mail)marsliydnb@163.com;王勇，(E-mail)wangyong119@ahau.edu.cn
更新日期/Last Update: 2020-07-14