Side-by-side Chinese-English

新城疫强毒株荧光定量RT-PCR检测技术的建立及应用

罗瑶瑶1,2 王静静1 吕艳1 赵云玲1 郑东霞1 魏润宇1,3 于松梅1 左媛媛1 张乐萃2 单虎2 刘华雷1,2 王志亮1

(1.中国动物卫生与流行病学中心, 青岛 266032)
(2.青岛农业大学动物医学院, 青岛 266109)
(3.扬州大学, 扬州 225009)
【创新点】基于国内流行毒株,建立了一种可检测所有流行强毒的荧光定量RT-PCR方法,结果证实本方法敏感性高、特异性强、重复性好、诊断准确性极好。通过对1974份临床样品检测,证实ROC曲线下面积为0.9861,诊断的敏感性和特异性分别为94.82 %、98.3 %,适用于新城疫大规模病原学主动监测和流行病学调查,具有显著的推广前景。

【摘要】基于国内流行的新城疫病毒强毒株F蛋白裂解位点分子特征设计特异性引物及Taqman探针,建立了一种可检测新城疫强毒株的一步法实时荧光定量RT-PCR方法。通过体外转录法制备cRNA标准品作为阳性模板制作标准曲线以及临床样品的检测绘制出ROC曲线,对诊断指标进行系统评价。结果显示:该方法的标准曲线为Y=-3.390X+38.23,相关系数R2为0.9999;灵敏度试验显示,该方法的最低检测限为2拷贝/μL,比常规RT-PCR高10倍;特异性试验显示该方法与常见禽病病毒无交叉反应;重复性试验的组内和组间的变异系数分别低于1%和1.5%。通过检测1 974份临床样品绘制ROC曲线显示,ROC曲线下面积为0.9861,当Youden Index为93.12时,cutoff值设为35,诊断的敏感性和特异性分别为94.82%、98.3%,与病毒分离方法的Kappa系数为0.919。本研究建立的方法敏感性高、特异性强、重复性好、诊断准确性极好,给实验室提供一种快速、实用的检测临床样品的方法。

【关键词】 新城疫病毒(NDV);强毒;荧光定量RT-PCR;ROC曲线;

【DOI】

【基金资助】 国家重点研发计划(项目号:2016YFD0500800),题目:家禽重要疫病诊断与检测新技术研究; 公益性行业(农业)科研专项(项目号:201303033),题目:鸡新城疫防控技术研究与示范;

Establishment and Application of Fluorescence Quantitative RT-PCR for Detection of the Velogenic Newcastle Disease Virus

LUO Yaoyao1,2 WANG Jingjing 1 LV Yan 1 ZHAO Yunling1 ZHENG Dongxia1 WEI Runyu1,3 YU Songmei1 ZUO Yuanyuan1 ZHANG Lecui2 SHAN Hu2 LIU Hualei1,2 WANG Zhiliang1

(1.China Animal Health and Epidemiology Center, Qingdao, China 266032)
(2.College of Veterinary Medicine, Qingdao Agricultural University, Qingdao, China 266109)
(3.Yangzhou University, Yangzhou, China 225009)
【Novelty】Based on the domestic epidemic strains, a fluorescence quantitative RT-PCR method was established to detecting velogenic NDVs. The results showed that the method was highly sensitive, specific, reproducible and accurate in diagnostic accuracy. It was proved that the area under ROC curve of 1,974 clinical samples was 0.9861. The sensitivity and specificity of diagnosis were 94.82 % and 98.3 % respectively. It was suitable for the active monitoring and epidemiological investigation of NDV on large-scale pathogens. It has a significant promotion prospect.

【Abstract】The primers and probe of a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were designed according to the characteristics of fusion protein cleavage sites of velogenic Newcastle disease viruses (NDVs). The analytical sensitivity of qRT-PCR was evaluated by standard curves using cRNA standards in vitro as positive controls. Analytical specificity was identified by detection of the common avian viruses and lentogenic NDV. Diagnostic sensitivity and specificity were validated based on receiver-operating characteristic (ROC) curves through detection of clinical samples. The limit of detection limit of this assay reached ≥ 2 copies and no cross-reaction was found. The coefficient of variation (CV) for intra-assay and inter-assay repeatability was < 1% and 1.5% respectively. The area under the ROC curve of 1974 clinical samples was 0.986 1. According to the Youden Index, the Cutoff value was 35. The sensitivity and specificity of this diagnosis was 94.82% and 98.3% respectively. The Kappa coefficient for isolation of the virus was 0.919. Our study demonstrated that a one-step qRT-PCR was highly sensitive, specific, reproducible and accurate, thereby providing a fast and practical method to detect the NDV in clinical samples.

【Keywords】 Newcastle disease virus (NDV); Virulent virus; Fluorescence quantitative RT-PCR; ROC curve;

【DOI】

【Funds】 National Key Research and Development Plan (2016YFD0500800); Public Welfare Industry (Agriculture) Research Project (201303033);

Download this article
    References

    [1] Aldous E W, Mynn J K, Irvine R M, Alexander D J, Brown I H. A molecular epidemiological investigation of avian paramyxovirus type 1 viruses isolated from game birds of the order Galliformes [J]. Avian Pathol, 2010, 39 (6): 519–524.

    [2] Ganar K, Das M, Raut A A, Mishra A, Kumar S. Emergence of a deviating genotype VI pigeon paramyxovirus type-1 isolated from India [J]. Arch Virol, 2017, 162 (7): 2169–2174.

    [3] Wang Z L, Liu H L. Newcastle disease [M] .2012, Beijing: China Agriculture Press (in Chinese).

    [4] Damena D, Fusaro A, Sombo M, Belaineh R, Heidari A, Kebede A, Kidane M, Chaka H. Characterization of Newcastle disease virus isolates obtained from outbreak cases in commercial chickens and wild pigeons in Ethiopia [J]. Springerplus, 2016, 5: 476–484.

    [5] Dimitrov K M, Ramey A M, Qiu X, Bahl J, Afonso CL. Temporal, geographic, and host distribution of avian paramyxovirus 1 (Newcastle disease virus) [J]. Infect Genet Evol, 2016, 39: 22–34.

    [6] Chong Y L, Kim O, Poss M. Lineage diversification of pigeon paramyxovirus effect re-emergence potential in chickens [J]. Virology, 2014, 462: 309–317.

    [7] Liu H L, Wang Z L. The history and current status of Newcastle disease [J]. Chinese Journal of Animal Health Inspection, 2015, 32 (6): 1–4 (in Chinese with English abstract).

    [8] Hu S L, Liu X F. The heredity and evolution of Newcastle disease virus [J]. Chinese Bulletin of Life Sciences, 2016, 28 (3): 295–302 (in Chinese with English abstract).

    [9] Liu H L, Lü Y, Wang J J, Zhao Y L. Distribution and characterization of emerged genotype XII Newcastle disease viruses in China [J]. China Animal Health Inspection, 2017, 34 (9): 1–4 (in Chinese with English abstract).

    [10] Xie Z, Xie L, Chen A, Liu J, Pang Y, Deng X, Xie Z, Fan Q. Complete genome sequence analysis of a Newcastle disease virus isolated from a wild egret [J]. J Virol, 2012, 86 (24): 13854–13855.

    [11] Fuller C M, Brodd L, Irvine R M, Alexander D J, Aldous E W. Development of an L gene real-time reverse transcription PCR assay for the detection of avian paramyxovirus type 1 RNA in clinical samples [J]. Arch Virol, 2010, 155 (6): 817–823.

    [12] Aguero M, Sanchez A, San Miguel E, Gomez-Tejedor C, Jimenez-Clavero M A. A real-time TaqMan RT-PCR method for neuraminidase type 1 (N1) gene detection of H5N1 Eurasian strains of avian influenza virus [J]. Avian Dis, 2007, 51 (1 Suppl): 378–381.

    [13] Bao J, Li L, Wang Z, Barrett T, Suo L, Zhao W, Liu Y, Liu C, Li J. Development of one-step real-time RT-PCR assay for detection and quantitation of peste des petits ruminants virus [J]. J Virol Methods, 2008, 148 (1–2): 232–236.

    [14] Huang Z J, Zhang D L, Shi J. Preparation of RNA standards for real-time fluorescence quantitative PCR [J]. Journal of Modern Laboratory Medicine, 2005, 20 (6): 18–21 (in Chinese with English abstract).

    [15] Beate M, Sharon K. Hietala, Liu M S, Lou L, Evan W S, Alex A A. High-throughput real-time RT-PCR assay to detect the exotic Newcastle disease virus during the California 2002–2003 outbreak [J]. J Vet Diagn Invest, 2005, 17: 124–132.

    [16] M. Greiner D P, R. D. Smith. Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests [J]. Pre Vet Med, 2000, 45: 23–41.

    [17] Laamiri N, Aouini R, Marnissi B, Ghram A, Hmila I. A multiplex real-time RT-PCR for simultaneous detection of four most common avian respiratory viruses [J]. Virology, 2018, 515: 29–37.

This Article

ISSN:1000-8721

CN: 11-1865/R

Vol 34, No. 04, Pages 541-549

June 2018

Downloads:1

Share
Article Outline

Novelty

Abstract

  • Materials and methods
  • Results
  • 7 Diagnostic performance evaluation
  • 8 Comparison test of different kits
  • Discussion
  • References