检测纽布病毒的Real-time RT-PCR方法的建立与应用

doi:10.11843/j.issn.0366-6964.2019.04.023

摘要/Abstract

摘要：

纽布病毒（nebovirus，NeV）是国内新发的犊牛腹泻病原，本试验目的是建立检测NeV的real-time RT-PCR方法。根据国内NeV流行株的聚合酶基因（RdRp）序列设计引物，通过反应条件和体系优化，成功建立基于TB Green检测NeV的real-time RT-PCR方法。结果显示：该方法在2.9×10¹~2.9×10⁹copies·μL^-1之间呈现良好的线性关系，线性相关系数R²=0.999 4，扩增效率为98%；该方法只特异性检出NeV，不检出常见无关病原；最低检测下限为29 copies·μL^-1；批内和批间的变异系数分别为0.89%~1.89%和0.83%~1.15%；该方法对临床样本的检出率高于文献报道的三种检测方法（P<0.05）。对2018年8-11月采自新疆和辽宁的135份奶犊牛腹泻样本中NeV的检出率为67.41%，场阳性率为100%（15/15）。所建方法的特异性和稳定性好、灵敏度高，为NeV的检测和流行病学调查提供了有力的手段。

Abstract:

Nebovirus (NeV) is an emerging causative agent of calf diarrhea in China, the aim of the study was to establish a real-time RT-PCR assay for detecting NeV. The TB Green real-time RT-PCR assay was successfully established through designing primers targeted to RdRp fragment of NeV strains in China and optimizing the reaction conditions and system. The Real-time RT-PCR assay showed a good linear relationship between 2.9×10¹ and 2.9×10⁹ copies·μL^-1, linear correlation coefficient R²=0.999 4, and amplification efficiency was 98%. This method only specifically detected NeV, and did not detect other unrelated pathogens; the minimum detection limit was 29 copies·μL^-1; the intra-and inter-coefficients of variation were 0.89%-1.89% and 0.83%-1.15%, respectively; comparing to other three reported RT-PCR assays, the assay in this study has a significant high detection rate for NeV in clinical samples (P<0.05). In 135 diarrhea samples of dairy cows from Xinjiang and Liaoing regions from August to November 2018, the NeV detection rate was 67.41%, and the farms positive rate was 100% (15/15). The assay for detecting NeV established in this study has a good specificity and stability, which provide an effective means for detection and epidemiological investigation of NeV.

中图分类号:

S852.659.6

郭紫晶, 何琪富, 汤承, 张斌, 岳华. 检测纽布病毒的Real-time RT-PCR方法的建立与应用[J]. 畜牧兽医学报, 2019, 50(4): 893-900.

GUO Zijing, HE Qifu, TANG Cheng, ZHANG Bin, YUE Hua. Establishment and Application of a Real-time RT-PCR Assay for Detecting Bovine Nebovirus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(4): 893-900.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2019.04.023

https://www.xmsyxb.com/CN/Y2019/V50/I4/893

参考文献

[1] CARTER M J, MILTON I D, MADELEY C R. Caliciviruses[J]. Rev Med Virol, 2010, 1(3):177-186.
[2] SMILEY J R, CHANG K O, HAYES J, et al. Characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus[J]. J Virol, 2002, 76(20):10089-10098.
[3] OTTO P H, CLARKE I N, LAMBDEN P R, et al. Infection of calves with bovine norovirus GⅢ. 1 strain Jena virus:an experimental model to study the pathogenesis of norovirus infection[J]. J Virol, 2011, 85(22):12013-12021.
[4] POURASGARI F, KAPLON J, SANCHOOLI A, et al. Molecular prevalence of bovine noroviruses and neboviruses in newborn calves in Iran[J]. Arch Virol, 2018, 163(5):1271-1277.
[5] TURAN T, I?IDAN H, ATASOY M O, et al. Detection and molecular analysis of bovine enteric norovirus and nebovirus in Turkey[J]. J Vet Res, 2018, 62(2):129-135.
[6] CANDIDO M, ALENCAR A L F, ALMEIDA-QUEIROZ S R, et al. First detection and molecular characterization of Nebovirus in Brazil[J]. Epidemiol Infect, 2016, 144(9):1876-1878.
[7] DI MARTINO B, DI PROFIO F, MARTELLA V, et al. Evidence for recombination in neboviruses[J]. Vet Microbiol, 2011, 153(3-4):367-372.
[8] GUO Z J, HE Q F, YUE H, et al. First detection of nebovirus and norovirus from cattle in China[J]. Arch Virol, 2018, 163(2):475-478.
[9] HASSINE-ZAAFRANE M, KAPLON J, SDIRI-LOULIZI K, et al. Molecular prevalence of bovine noroviruses and neboviruses detected in central-eastern Tunisia[J]. Arch Virol, 2012, 157(8):1599-1604.
[10] KAPLON J, GUENAU E, ASDRUBAL P, et al. Possible novel nebovirus genotype in cattle, France[J]. Emerg Infect Dis, 2011, 17(6):1120-1123.
[11] OLIVER S L, ASOBAYIRE E, DASTJERDI A M, et al. Genomic characterization of the unclassified bovine enteric virus Newbury agent-1(Newbury1) endorses a new genus in the family Caliciviridae[J]. Virology, 2006, 350(1):240-250.
[12] PARK S I, JEONG C, PARK S J, et al. Molecular detection and characterization of unclassified bovine enteric caliciviruses in South Korea[J]. Vet Microbiol, 2008, 130(3-4):371-379.
[13] PANKOVICS P, BOROS A, NEMES C, et al. First detection of nebovirus (Caliciviridae) in faecal sample of diarrhoeic calf in Hungary[J]. Magy Allatorvosok Lapja, 2013, 135:12-17.
[14] WOODE G N, BRIDGER J C. Isolation of small viruses resembling astroviruses and caliciviruses from acute enteritis of calves[J]. J Med Microbiol, 1978, 11(4):441-452.
[15] BRIDGER J C, HALL G A, BROWN J F. Characterization of a calici-like virus (Newbury agent) found in association with astrovirus in bovine diarrhea[J]. Infect Immun, 1984, 43(1):133-138.
[16] CHO Y I, HAN J I, WANG C, et al. Case-control study of microbiological etiology associated with calf diarrhea[J]. Vet Microbiol, 2013, 166(3-4):375-385.
[17] GUO Z J, HE Q F, ZHANG B, et al. Detection and molecular characteristics of neboviruses in dairy cows in China[J]. J Gen Virol, 2018, 100(1):35-45.
[18] GUO Z J, HE Q F, YUE H, et al. Genomic characterization of a RdRp-recombinat nebovirus strain with a novel VP1 genotype[J]. Virus Res, 2018, 251:6-13.
[19] LEE J H, CHUNG M S, KIM K H. Structure and function of caliciviral RNA polymerases[J]. Viruses, 2017, 9(11):329.
[20] PARK S I, PARK D H, SAIF L J, et al. Development of SYBR Green real-time RT-PCR for rapid detection, quantitation and diagnosis of unclassified bovine enteric calicivirus[J]. J Virol Methods, 2009, 159(1):64-68.
[21] PARK S J, JEONG C, YOON S S, et al. Detection and characterization of bovine coronaviruses in fecal specimens of adult cattle with diarrhea during the warmer seasons[J]. J Clin Microbiol, 2006, 44(9):3178-3188.
[22] SMILEY J R, HOET A E, TRÅVÉN M, et al. Reverse transcription-PCR assays for detection of bovine enteric caliciviruses (BEC) and analysis of the genetic relationships among bec and human caliciviruses[J]. J Clin Microbiol, 2003, 41(7):3089-3099.
[23] GOMEZ D E, WEESE J S. Viral enteritis in calves[J]. Can Vet J, 2017, 58(12):1267-1274.
[24] DENG M L, JI S K, FEI W T, et al. Prevalence study and genetic typing of bovine viral diarrhea virus (BVDV) in four bovine species in China[J]. PLoS One, 2015, 10(4):e0121718.
[25] 周芳,岳华,张斌,等. 牦牛轮状病毒VP6基因序列分析及RT-PCR检测方法的建立与应用[J]. 畜牧兽医学报, 2016, 47(7):1465-1473.
ZHOU F, YUE H, ZHANG B, et al. Establishment and application of an RT-PCR assay for yak rotavirus based on the sequence analysis of yak rotavirus VP6 gene[J]. Acta Veterinaria et Zootechnica Sinica, 2016, 47(7):1465-1473. (in Chinese)
[26] 何琪富, 郭紫晶,李然,等. 牛冠状病毒RT-PCR检测方法的建立及应用[J]. 畜牧兽医学报, 2018, 49(10):2292-2298.
HE Q F, GUO Z J, LI R, et al. Establishment and application of a RT-PCR assay for detecting bovine coronavirus[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(10):2292-2298. (in Chinese)
[27] 陈新诺, 肖敏,阮文强,等. 川藏地区牦牛牛病毒性腹泻病毒分子流行病学调查及分离鉴定[J]. 畜牧兽医学报, 2018, 49(3):606-613.
CHEN X N, XIAO M, RUAN W Q, et al. Molecular epidemiological investigation and isolation of bovine viral diarrhea virus in yak in Sichuan-Tibet Plateau region[J]. Acta Veterinaria et Zootechnica Sinica, 2018, 49(3):606-613. (in Chinese)
[28] DI FELICE E, MAUROY A, DAL POZZO F, et al. Bovine noroviruses:A missing component of calf diarrhoea diagnosis[J]. Vet J, 2016, 207:53-62.
[29] GÜLAÇTⅡ, I?IDAN H, SÖZDUTMAZI. Detection of bovine torovirus in fecal specimens from calves with diarrhea in Turkey[J]. Arch Virol, 2014, 159(7):1623-1627.
[30] GUO Z J, HE Q F, TANG C, et al. Identification and genomic characterization of a novel CRESS DNA virus from a calf with severe hemorrhagic enteritis in China[J]. Virus Res, 2018, 255:141-146.
[31] CHEN X, ZHANG B, YUE H, et al. A novel astrovirus species in the gut of yaks with diarrhoea in the Qinghai-Tibetan Plateau, 2013[J]. J Gen Virol, 2015, 96(12):3672-3680.