Scientia Agricultura Sinica ›› 2012, Vol. 45 ›› Issue (21): 4492-4500.doi: 10.3864/j.issn.0578-1752.2012.21.018

• VETERINARY SCIENCE • Previous Articles     Next Articles

Fluorescence Quantitative RT-PCR Assay for Detection of Tembusu Virus

 YU  Chun-Mei, DIAO  You-Xiang, TANG  Yi, CUI  Jing-Teng, GAO  Xu-Hui, ZHANG  Ying, JU  Xiao-Jun, WU  Li-Li   

  1. 1.山东农业大学动物科技学院  山东泰安 271018
  • Received:2012-03-23 Online:2012-11-01 Published:2012-09-27

PDF

1066

Cited

13

Abstract: 【Objective】The objective of the study is to establish a method for detecting Tembusu virus by SYBR GreenⅠ relative fluorescence quantitative RT-PCR. 【Method】 Special primers based on Tembusu virus NS5 and E gene were designed and a pair of primers of house-keeping gene β-actin was chosen. Then these amplified fragments were cloned into pMD18-T. Using the plasmids NS5-pMD18-T, E-pMD18-T and β-actin-pMD18-T as standard products, a real-time quantitative reverse transcription-polymerase chain reaction ( RT-PCR) was performed to construct the standard curves of NS5, E and β-actin gene and detect the sensitivity, specificity and repeatability. 【Result】 The results showed a precise linear relationship with a correlation coefficient of R2＞0.99. The detection limits was 10 copies of DNA plasmid reaction. The amplification curve showing a single peak could only been detected for Tembusu virus. The variation coefficient was less than 0.5% by within and between the group of repeatability tests. The clinical samples were detected 3 times by this method, and all results were positive.【Conclusion】 The developed real-time PCR assay was highly specific, sensitive, and reproducible and could be an available tool for diagnosis and monitoring of Tembusu virus in duck farms.

Key words:

[1]曹贞贞, 张  存, 黄  瑜, 刁有祥, 叶伟成, 刘月焕, 韩婧文, 马国明, 张冬冬. 鸭出血性卵巢炎的初步研究. 中国兽医杂志, 2010, (46)12: 4-6.

Cao Z Z, Zhang C, Huang Y, Diao Y X, Ye W C, Liu Y H, Han J W, Ma G M, Zhang D D. Preliminary studies on duck hemorrhagic ovaritis. Chinese Journal of Veterinary Medicine, 2010, 46(12): 4-6. (in Chinese)

[2]Tang Y, Diao Y X, Gao X H. Analysis of the complete genome of  tembusu virus, a flavivirus isolated from ducks in China. Transboundary and Emerging Diseases, 2011(9): 1-10.

[3]Su J L, Li S, Hu X D, Yu X L, Wang Y Y, Liu P P, Lu X S, Zhang G Z, Hu X Y, Liu D, Li X X, Su W L, Lu H. Duck egg-drop syndrome caused by BYD virus, a new tembusu-related flavivirus. Plos One, 2011, 6 (3): 18106

[4]Yan P X, Zhao Y S, Zhan X G, Xu D W, Dai X G, Teng Q Y, Yan L P, Zhou J W, Ji X W, Zhang S M, Liu G Q, Zhou Y J, Kawaoka Y, Tong G Z, Li Z. An infectious disease of ducks caused by a newly emerged Tembusu virus strain in mainland China. Virology, 2011, 417(1): 1-8.

[5]林 健, 韩春华, 陈华林, 蒋桃珍, 梁 武, 何平有, 杨保收, 王 英, 韩婧雯, 刘东艳, 潘 洁, 段会娟, 丁佩佩, 胡胜强, 刘月焕. 鸭出血性卵巢炎实验感染模型的建立. 中国农业科学, 2011, 44(10): 2161-2167.

Lin J, Han C H, Chen H L, Jiang T Z, Liang W, He P Y, Yang B S, Wang Y, Han J W, Liu D Y, Pan J, Duan H J, Ding P P, Hu S Q, Liu Y H. Establishment of experimental infection model of duck hemorrhagic ovaritis. Scientia Agricultura Sinica, 2011, 44(10): 2161-2167. (in Chinese)

[6]颜丕熙, 李国新, 吴晓刚, 闫丽萍, 滕巧泱, 李泽君. 应用套式RT-PCR快速检测鸭坦布苏病毒. 中国动物传染病学报, 2011, 19(3): 34-37.

Yan P X, Li G X,  Wu X G, Yan L P, Teng Q Y, Li Z J. Rapid identification of duck tembusu virus by the nested RT-PCR. Chinese Journal of Animal Infectious Diseases, 2011, 19(3): 34-37. (in Chinese)

[7]唐　熠, 刁有祥, 高绪慧, 于春梅, 张大丙, 岳澄滨. 鸭黄病毒感染逆转录半套式PCR检测方法的建立. 中国兽医学报, 2012, 32(4): 517-520.

Tang Y, Diao Y X, Gao X H, Yu C M, Zhang D B, Yue C B. Development of a semi-nested RT-PCR assay for detection of duck flavivirus. Chinese Journal of Veterinary Science, 2012, 32(4): 517-520. (in Chinese)

[8]Wang Y L, Yuan X Y, Li Y F, Yu K X, Yang J X, Xu H Y, Zhang Y X, Yu K Z, Liao M, Qin Z M. Rapid detection of newly isolated tembusu-related flavivirus by reverse-transcription loop-mediated isothermal amplification assay. Virology Journal, 2011, 8: 553.

[9]Tang Y, Diao Y X, Yu C M. Rapid detection of tembusu virus by reverse-transcription, loop-mediated isothermal amplification (RT-LAMP). Transboundary and Emerging Diseases, 2011(6): 1-9.

[10]姬希文, 闫丽萍, 颜丕熙, 李国新, 张七斤, 李泽君. 鸭坦布苏病毒抗体间接ELISA 检测方法的建立. 中国预防兽医学报, 2011, 33(8): 630-634.

Ji X W, Yan L P, Yan P X, Li G X, Zhang Q Q, Li Z J. Establishment of an indirect ELISA for detection of antibody against duck tembusu virus. Chinese Journal of Preventive Veterinary Medicine, 2011, 33(8): 630-634. (in Chinese)

[11]高绪慧, 刁有祥, 唐  熠, 于春梅, 张大丙, 岳澄滨. 探针检测鸭黄病毒的地高辛标记DNA的制备与应用. 中国兽医学报, 2012, 32(4): 525-528.

Gao X H, Diao Y X, Tang T,  Yu C M, Zhang D B, Yue C B. Preparation of digoxigenin-labeled DNA probe for detection of duck flavivirus and its application. Chinese Journal of Veterinary Science, 2012, 32(4): 525-528. (in Chinese)

[12]DeGraves F J, Gao D Y, Kaltenboeck B. High-sensitivity quantitative PCR platform. Biotechniques, 2003, 34: 106-115.

[13]童桂香, 谢芝勋, 黄  琦, 文艳玲, 谢丽基, 庞耀山, 刘加波, 邓显文. 禽呼肠孤病毒实时荧光定量 RT-PCR检测方法的建立. 中国兽医科学, 2007, 37(9): 767-771.

Tong G X, Xie Z X, Huang Q, Wen Y L, Xie L J, Pang Y S, Liu J B, Deng X W. Development of real time fluorescent quantitative RT-PCR for detection of avian reovirus. Veterinary Science in China, 2007, 37(9): 767-771. (in Chinese)

[14]董  浩, 徐楠楠. 鹅细小病毒荧光定量PCR检测方法的建立及应用. 中国兽药杂志, 2011, 45(9): 12-14.

Dong H, Xu N N. Establishment and application of fluorescence quantitative PCR for identification of goose parvovirus. Chinese Journal of Veterinary Drug, 2011, 45( 9): 12-14. (in Chinese)

[15]熊文婕, 谢芝勋, 唐  熠, 谢丽基, 刘加波, 庞耀姗, 邓显文, 谢志勤. SYBR GreenⅠ实时荧光定量RT－PCR检测禽呼肠孤病毒δC和δNS基因方法的建立. 广西农业科学, 2010, 41(4): 366-370.

Xiong W J, Xie Z X, Tang Y, Xie L J, Liu J B, Pang Y S, Deng X W, Xie Z Q. Detection of avian reovirus δC and δNS genes by SYBR GreenⅠrelative quantitative RT-PCR assay. Guangxi Agricultural Sciences, 2010, 41(4): 366-370. (in Chinese)

[16]张大丙. 鸭出血性卵巢炎的研究进展. 中国家禽, 2011, 33(14): 1-2.

Zhang D B. Recent studies of duck hemorrhagic ovaritis. China Poultry, 2011, 33(14): 1-2. (in Chinese)

[17]陈  坚, 刘业兵, 张志刚, 姜晓晨, 张晓杰. SYBR GreenⅠ 实时定量PCR方法检测猪细小病毒. 中国兽医杂志, 2011, 47(8): 6-7.

Chen J, Liu Y B, Zhang Z G, Jang X C, Zhang X J. Establishment of SYBR GreenⅠreal time PCR assay for the detection of porcine parvovirus. Chinese Journal of Veterinary Medicine, 2011, 47(8): 6-7. (in Chinese)

[18]Yan L P, Yan P X, Zhou J W, Teng Q Y, Li Z J. Establishing a TaqMan-based real-time PCR assay for the rapid detection and quantification of the newly emerged duck tembusu virus. Virology Journal, 2011, 8: 464-471.

[19]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. 中国病原生物学杂志, 2008, 3(8): 561-563.

Zhou X J, Zhu H M, Qiu L. Establishment of RT-PCR and SYBR Green Real-time PCR for detection of flavivirus in mosquito. Journal of Pathogen Biology, 2008, 3(8): 561-563. (in Chinese)

[20]周云雷, 魏飞龙, 李 健, 张小华, 蒋红霞. 鸡毒支原体实时荧光定量PCR 检测方法的建立. 中国农业科学, 2011, 44(11): 2371-2378.

Zhou Y L, Wei F L, Li J, Zhang X H, Jiang H X. Development of real-time fluorescent quantitative PCR method for detection of mycoplasma gallisepticum. Scientia Agricultura Sinica, 2011, 44(11): 2371-2378. (in Chinese)

[21]罗  婵, 王志强, 公方强, 石德顺. SYBR Green 实时荧光定量PCR检测水牛体细胞组蛋白乙酰化相关基因mRNA表达. 中国兽医学报, 2009, 29(2): 233-237.

Luo C, Wang Z Q, Gong F Q, Shi D S. Detection of histone acetylation related gene expression in buffalo somatic cells by SYBR Green Ñrea-l time f luorescence quantitative PCR. Chinese Journal of Veterinary Science, 2009, 29(2): 233-237. (in Chinese)

[22]罗招凡, 丁鹤林, 刘建伟. SYBR GreenⅠ荧光定量PCR检测西尼罗病毒. 中华医院感染学杂志, 2007, 17(8): 1031-1034.

Luo Z F, Ding H L, Liu J W. Detection of west nile virus using SYBR GreenⅠfluorescent quantitative PCR assay. Chinese Journal of Nosocomiology, 2007, 17(8): 1031-1034. (in Chinese)

[23]Seo K S, Park J Y, Terman D S, Bohach G A. A quantitative real time PCR method to analyze T cell receptor Vb subgroup expansion by staphylococcal superantigens. Journal of Translational Medicine, 2010, 8(2): 1-9.

[24]Jian B, Liu B, Bi Y R, Hou W S, Wu C X, Han T F. Validation of internal control for gene expression study in soybean by quantitative real-time PCR. BMC Molecular Biology, 2008, 59(9): 1-14.

[25]Scheurer M E, Dillon L M, Chen Z, Follen M, Adler-Storthz K. Absolute quantitative real-time polymerase chain reaction for the measurement of human papillomavirus E7 mRNA in cervical cytobrush specimens. Infectious Agents and Cancer, 2007, 2(8): 1-9.
[1] YE MeJin, WU Lei, MD NAHIBUZZAMAN Lohani, YIN Li, HU XinRong, LIU YaXi, JIANG YunFeng, CHEN GuoYue, PU ZhiEn, LI Yang, LI Ting, ZOU YaYa, WU JiaYi, MA Jian. Genome-Wide Association Study-Based Identification of Loci Controlling Mature Embryo Size in Chinese Wheat Landraces and Their Genetic Effects Analysis [J]. Scientia Agricultura Sinica, 2026, 59(6): 1157-1171.
[2] WANG JiaNuo, CHEN GuiPing, LI Pan, WANG LiPing, NAN YunYou, HE Wei, FAN ZhiLong, HU FaLong, CHAI Qiang, YIN Wen, ZHAO LiaoHao. Photo-Physiological Mechanism at Grain Filling Stage of No-Tillage with Plastic Re-Mulching to Increase Maize Yield in Oasis Irrigation Areas [J]. Scientia Agricultura Sinica, 2026, 59(6): 1189-1202.
[3] WANG YongSheng, NIU Li, WANG ChangJie, MA LiHua, LIAN XiaoXiao, MENG YaXiong, MA XiaoLe, YAO LiRong, ZHANG Hong, YANG Ke, LI BaoChun, WANG HuaJun, SI ErJing, WANG JunCheng. Genome-Wide Association Study and Candidate Gene Identification for Thousand Grain Weight in Winter Wheat [J]. Scientia Agricultura Sinica, 2026, 59(3): 499-514.
[4] SHEN YingZi, LI DuoJiao, JIANG Li, HU XingRong, CHEN Bin, ZHENG ZhaiSheng, YUAN MingAn. Functional Analysis of the CsGPDH Gene in Seed Oil Accumulation of Tea Plant [J]. Scientia Agricultura Sinica, 2026, 59(2): 402-412.
[5] LI YunLi, DIAO DengChao, LIU YaRui, SUN YuChen, MENG XiangYu, WU ChenFang, WANG Yu, WU JianHui, LI ChunLian, ZENG QingDong, HAN DeJun, ZHENG WeiJun. Genome-Wide Association Study of Heat Tolerance at Seedling Stage in A Wheat Natural Population [J]. Scientia Agricultura Sinica, 2025, 58(9): 1663-1683.
[6] ZOU XiaoWei, XIA Lei, ZHU XiaoMin, SUN Hui, ZHOU Qi, QI Ji, ZHANG YaFeng, ZHENG Yan, JIANG ZhaoYuan. Analysis of Disease Resistance Induced by Ustilago maydis Strain with Overexpressed UM01240 Based on Transcriptome Sequencing [J]. Scientia Agricultura Sinica, 2025, 58(6): 1116-1130.
[7] GAO YanHao, WANG TingTing, BAI WeiWei, DU XingJie, LIU Xian, QIN BenYuan, FU Tong, SUN Yu, GAO TengYun, ZHANG TianLiu. The Combination of Lipidome and Transcriptome Revealed the Differential Expression Patterns of Lipid Characteristics in Different Muscle Tissues for Nanyang Cattle [J]. Scientia Agricultura Sinica, 2025, 58(6): 1239-1258.
[8] ZHOU GuangFei, MA Liang, MA Lu, ZHANG ShuYu, ZHANG HuiMin, SONG XuDong, ZHANG ZhenLiang, LU HuHua, HAO DeRong, MAO YuXiang, XUE Lin, CHEN GuoQing. Genome-Wide Association Study of Husk Traits in Maize [J]. Scientia Agricultura Sinica, 2025, 58(3): 431-442.
[9] ZHAO DongLan, MA JuKui, XIAO ShiZhuo, ZHOU ZhiLin, ZHAO LingXiao, WANG Jie, DAI XiBin, SUN HouJun, CAO QingHe. QTL Analysis for Resistance to Stem Nematode Disease in Sweetpotato [J]. Scientia Agricultura Sinica, 2025, 58(17): 3389-3399.
[10] LIU ChenXi, ZHAO BingBing, SHI ZhiBin, WANG ShiDa, WANG JingFei. Construction of Infectious Clones for Canine Parvovirus CPV-2c SX-LC Strain and Virus Rescue [J]. Scientia Agricultura Sinica, 2025, 58(17): 3561-3570.
[11] DONG Xue, CHEN MengQiu, SHAO Jin, WU XueYou, TANG PeiAn. Construction of a Differential Gene Expression and Quality Regulation Network in Stored Rice Grain Using WGCNA [J]. Scientia Agricultura Sinica, 2025, 58(14): 2885-2903.
[12] XIE HuiHui, YANG QiuHua, LI WenLi, ZHU JinCheng, LI HuiXia, ZHANG Feng. Identification of Wild Potato Introgression Lines Resistant to Southern Root-Knot Nematode [J]. Scientia Agricultura Sinica, 2025, 58(14): 2924-2932.
[13] LI Ning, GAO LiFeng, HUANG Xin, SHI HuaWei, YANG JinWen, SHI YuGang, CHEN Ming, JIA JiZeng, SUN DaiZhen. Screening of Wheat Varieties with Low Nitrogen Tolerance and Genome-Wide Association Studies of Low Nitrogen Stress Tolerance Index [J]. Scientia Agricultura Sinica, 2025, 58(13): 2487-2503.
[14] DENG LiCheng, LI Cheng, HE Lei, AN HongQiang, WANG CaiLin, ZHANG YaDong, ZHAO ChangJiang, LU Kai. Physiological Characteristics in Response to Salt Stress and Allelic Variation and Expression of Salt-Responsive Genes in Seedling Stage of Nangeng Rice Varieties with Salt-Tolerance Ability [J]. Scientia Agricultura Sinica, 2025, 58(12): 2275-2290.
[15] WEN Jin, NING YanFang, QIN Xin, LIU Yuan, ZHANG XiaoLing, ZHU YongHong, TIAN ShiMin, MA YanBin. Resistance Evaluation and Genetic Stability Analysis of Insect- Resistant and Glyphosate-Tolerant Transgenic Cotton Lines [J]. Scientia Agricultura Sinica, 2025, 58(12): 2291-2302.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd