新型鸭呼肠孤病毒一步法TaqMan-MGB荧光定量RT-PCR检测方法的建立与应用

doi:10.3969/j.issn.1004-1524.2020.04.02

浙江农业学报 ›› 2020, Vol. 32 ›› Issue (4): 571-576.DOI: 10.3969/j.issn.1004-1524.2020.04.02

新型鸭呼肠孤病毒一步法TaqMan-MGB荧光定量RT-PCR检测方法的建立与应用

云涛, 华炯钢, 叶伟成, 倪征, 陈柳, 张存^*

浙江省农业科学院畜牧兽医研究所,浙江杭州 310021

收稿日期:2019-11-18 出版日期:2020-04-25 发布日期:2020-04-26
通讯作者: *张存,E-mail: zhangcun@aliyun.com
作者简介:云涛(1978—),男,浙江杭州人,博士,副研究员,从事禽病毒病分子生物学研究。E-mail: yt-t@163.com
基金资助:
浙江省重大科技专项重点农业项目(2015C02012); 浙江省重点研发计划(2019C02052)

Development and application of a one-step real-time TaqMan-MGB RT-PCR assay for detection of novel duck reovirus

YUN Tao, HUA Jionggang, YE Weicheng, NI Zheng, CHEN Liu, ZHANG Cun^*

Institute of Animal Husbandry and Veterinary Sciences, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2019-11-18 Online:2020-04-25 Published:2020-04-26

摘要/Abstract

摘要： 为建立一种快速、敏感和特异性检测新型鸭呼肠孤病毒(novel duck reovirus,NDRV)的方法,本研究根据GenBank数据库中NDRV S3基因保守序列设计1对特异性引物和1条MGB荧光探针,建立了一种检测NDRV的一步法MGB荧光定量RT-PCR方法,对其特异性、敏感性和重复性进行检验,并与普通RT-PCR进行比较。结果显示,扩增标准曲线相关系数为0.999,扩增产物的熔解曲线仅出现单特异峰。该方法对经典鸭呼肠孤病毒、H9N2禽流感病毒、鸭坦布苏病毒、A型鸭肝炎病毒、鸭新城疫病毒、鸭瘟病毒及番鸭细小病毒均未检测到信号,对NDRV的最小检出量为10拷贝·μL^-1。组内和组间变异系数均小于2%,重复性好。此外,利用该方法对239份疑似新型呼肠孤病毒病样品进行检测,常规RT-PCR检测时有75份为阳性,一步法TaqMan-MGB荧光定量RT-PCR方法检测时有100份为阳性,而且常规RT-PCR检测出的阳性样品用一步法TaqMan-MGB荧光定量RT-PCR检测时均为阳性,符合率为100%。该检测方法的建立为NDRV早期快速检测及定量分析提供新的方法。

关键词: 新型鸭呼肠孤病毒, MGB探针, 荧光定量RT-PCR, 检测方法

Abstract: To develop a fast, sensitive, specific TaqMan MGB probe real-time fluorescent quantitative PCR assay for detecting novel duck reovirus (NDRV), a pair of specific primers and a MGB probe were designed according to the conserved region of S3 segment of the NDRV genome. The results showed that recombinant plasmids were used to generate standard curve with correlation coefficient (R²) of 0.999, and the melting curve showed one specific peak. No cross-reaction was detected for classical muscovy duck reovirus (CDRV), avian influenza virus (AIV), duck Temubusu virus (DTMUV), duck hepatitis A virus (DHV-1), Newcastle diseases virus (NDV), duck plague virus (DPV) and muscovy duck parvovirus (MDPV). The limit detection of real-time RT-PCR was about 10 copies·μL^-1 for the target gene. The reproducibility tests in inter-assay and intra-assay indicated that the coefficients of variation were less than 2%. In addition, a collection of 239 clinical samples from Zhejiang during the year 2011-2015 were detected by the assay and conventional RT-PCR, respectively. The results showed that 75 samples were positive in the conventional RT-PCR test while 100 samples were positive in the one-step MGB probe real-time RT-PCR test. All the positive samples detected by conventional RT-PCR were positive by one-step MGB probe RT-PCR test, and the coincidence rate was 100%. The method established in this study could be useful for earlier rapid laboratory diagnosis and quantitative analysis of the infection of NDRV.

Key words: novel duck reovirus, MGB probe, fluorescent quantitative RT-PCR, diagnosis method

中图分类号:

S852.65

云涛, 华炯钢, 叶伟成, 倪征, 陈柳, 张存. 新型鸭呼肠孤病毒一步法TaqMan-MGB荧光定量RT-PCR检测方法的建立与应用[J]. 浙江农业学报, 2020, 32(4): 571-576.

YUN Tao, HUA Jionggang, YE Weicheng, NI Zheng, CHEN Liu, ZHANG Cun. Development and application of a one-step real-time TaqMan-MGB RT-PCR assay for detection of novel duck reovirus[J]. , 2020, 32(4): 571-576.

参考文献

[1] 吴宝成, 陈家祥, 姚金水, 等. 番鸭呼肠孤病毒的分离与鉴定[J]. 福建农业大学学报, 2001, 30(2): 227-230.
WU B C, CHEN J X, YAO J S, et al.Isolation and identification of muscovy duck reovirus[J]. Journal of Fujian Agricultural University(Natural Science), 2001, 30(2): 227-230.(in Chinese with English abstract)
[2] 陈少莺, 陈仕龙, 林锋强, 等. 新型鸭呼肠孤病毒的分离与鉴定[J]. 病毒学报, 2012, 28(3): 224-230.
CHEN S Y, CHEN S L, LIN F Q, et al.The isolation and identification of novel duck reovirus[J]. Chinese Journal of Virology, 2012, 28(3): 224-230.(in Chinese with English abstract)
[3] MA G, WANG D, SHI J, et al.Complete genomic sequence of a reovirus isolate from Pekin ducklings in China[J]. Journal of Virology, 2012, 86(23): 13137.
[4] YUN T, YU B, NI Z, et al.Genomic characteristics of a novel reovirus from muscovy duckling in China[J]. Veterinary Microbiology, 2014, 168(2/3/4): 261-271.
[5] YUN T, YU B, NI Z, et al.Isolation and genomic characterization of a classical muscovy duck reovirus isolated in Zhejiang, China[J]. Infection, Genetics and Evolution, 2013, 20: 444-453.
[6] WANG D, SHI J J, YUAN Y, et al.Complete sequence of a reovirus associated with necrotic focus formation in the liver and spleen of muscovy ducklings[J]. Veterinary Microbiology, 2013, 166(1/2): 109-122.
[7] 黄瑜, 傅光华, 施少华, 等. 新致病型鸭呼肠孤病毒的分离鉴定[J]. 中国兽医杂志, 2009, 45(12): 29-31.
HUANG Y, FU G H, SHI S H, et al.Isolation and identification of a new muscovy duck reovirus[J]. Chinese Journal of Veterinary Medicine, 2009, 45(12): 29-31.(in Chinese)
[8] 王光锋, 王永坤, 王建业, 等. 一株鹅源呼肠孤病毒的分离与鉴定[J]. 中国家禽, 2003, 25(15): 8-10.
WANG G F, WANG Y K, WANG J Y, et al.Isolation and identification of one goose reovirus strain[J]. China Poultry, 2003, 25(15): 8-10.(in Chinese with English abstract)
[9] 陈仕龙, 陈少莺, 程晓霞, 等. 3种禽类呼肠孤病毒血清学相关性及致细胞病变差异分析[J]. 畜牧兽医学报, 2011, 42(4): 533-537.
CHEN S L, CHEN S Y, CHENG X X, et al.The comparison of serologic relativity and CPE types of 3 avian reovirus strains induced different diseases[J]. Chinese Journal of Animal and Veterinary Sciences, 2011, 42(4): 533-537.(in Chinese with English abstract)
[10] 陆新浩, 刘鸿, 陈秋英, 等. 一种番鸭“新肝病”在浙江的流行特点分析[J]. 中国家禽, 2011, 33(15): 49-50.
LU X H, LIU H, CHEN Q Y, et al.An analysis of the epidemic characteristics of “new liver disease” of muscovy duck in zhejiang province[J]. China Poultry, 2011, 33(15): 49-50.(in Chinese)
[11] 袁远华, 吴志新, 王俊峰, 等. 新型鸭呼肠孤病毒SYBR Green Ⅰ实时荧光定量RT-PCR检测方法的建立[J]. 中国预防兽医学报, 2013, 35(9): 738-741.
YUAN Y H, WU Z X, WANG J F, et al.Development of a SYBR Green Ⅰ real-time RT-PCR assay for rapid diagnosis of new-type duck reovirus[J]. Chinese Journal of Preventive Veterinary Medicine, 2013, 35(9): 738-741.(in Chinese with English abstract)
[12] 丁明洋, 戚伟强, 陈宗艳, 等. 一种新型鸭呼肠孤病毒SYBR GreenⅡ荧光定量PCR方法的建立[J]. 中国动物传染病学报, 2016, 24(1): 7-14.
DING M Y, QI W Q, CHEN Z Y, et al.Detection of novel duck reovirus using SYBR green ⅱ fluorescent quantitative pcr assay[J]. Chinese Journal of Animal Infectious Diseases, 2016, 24(1): 7-14.(in Chinese with English abstract)
[13] 云涛, 张存, 华炯钢, 等. 鸭呼肠孤病毒基因Ⅰ型与Ⅱ型鉴别诊断RT-PCR方法的建立与应用[J]. 农业生物技术学报, 2016, 24(12): 1964-1972.
YUN T, ZHANG C, HUA J G, et al.Establishment and application of RT-PCR for identification and diagnosis of duck reovirus genotype Ⅰ and Ⅱ[J]. Journal of Agricultural Biotechnology, 2016, 24(12): 1964-1972.(in Chinese with English abstract)
[14] CHEN Z Y, ZHU Y Q, LI C F, et al.Outbreak-associated novel duck reovirus, China, 2011[J]. Emerging Infectious Diseases, 2012, 18(7): 1209-1211.
[15] YUN T, YE W, NI Z, et al.Complete genomic sequence of goose-origin reovirus from China[J]. Journal of Virology, 2012, 86(23): 13143.
[16] KUNTZ-SIMON G, LE GALL-RECULÉ G, DE BOISSÉSON C, et al. Muscovy duck reovirus σC protein is atypically encoded by the smallest genome segment[J]. Journal of General Virology, 2002, 83(5): 1189-1200.
[17] MARTÍNEZ-COSTAS J, GRANDE A, VARELA R, et al. Protein architecture of avian reovirus S1133 and identification of the cell attachment protein[J]. Journal of Virology, 1997, 71(1): 59-64.
[18] SHAPOURI M R S, KANE M, LETARTE M, et al. Cloning, sequencing and expression of the S1 gene of avian reovirus[J]. Journal of General Virology, 1995, 76(6): 1515-1520.
[19] YUN T, NI Z, HUA J G, et al.Development of a one-step real-time RT-PCR assay using a minor-groove-binding probe for the detection of duck Tembusu virus[J]. Journal of Virological Methods, 2012, 181(2): 148-154.
[20] WANG S Q, WANG X H, CHEN S H, et al.A new fluorescent quantitative polymerase chain reaction technique[J]. Analytical Biochemistry, 2002, 309(2): 206-211.
[21] AFONINA I, KUTYAVIN I, LUKHTANOV E, et al.Sequence-specific arrest of primer extension on single-stranded DNA by an oligonucleotide-minor groove binder conjugate[J]. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93(8): 3199-3204.

新型鸭呼肠孤病毒一步法TaqMan-MGB荧光定量RT-PCR检测方法的建立与应用

Development and application of a one-step real-time TaqMan-MGB RT-PCR assay for detection of novel duck reovirus

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