检测施马伦贝格病毒核酸的荧光定量RT-PCR方法

doi:10.11843/j.issn.0366-6964.2014.11.012

Abstract

Abstract:

In the present study，a pair of primers and a TaqMan probe were designed based on the S-segment sequence of Schmallenberg virus (SBV)，and a real-time quantitative reverse transcription-PCR (RT-qPCR) for the detection of SBV nucleic acid was established.After optimization of the reaction system and condition，the method presented a good linear relationship with the RNA template ranging from 10¹ to 10⁷ 50% tissue culture infectious dose (TCID₅₀) SBV.By using SBV nucleic acid-positive samples (n=140)，SBV nucleic acid-negative samples (n=132)，and RNA samples (n=16) of related viruses within the Simbu serogroup，the sensitivity，specificity and reproducibility of the RT-qPCR were validated.All the samples used for validation were prepared and preserved by the Friedrich-Loeffler-Institut (FLI) of Germany.The results demonstrated that the developed RT-qPCR had a similar sensitivity with that of FLI and the concordance rate between the two assays was 96.4% (135/140)，that the RT-qPCR could sensitively detect 1 TCID₅₀ SBV RNA and it had a good reproducibility，and that the RT-qPCR showed no cross-reactivity with related viruses within the Simbu serogroup except for Douglas virus.The RT-qPCR was further used to detect the RNA samples extracted from sheep sera which were collected from a breeding farm located in Inner Mongolia (n=47) and from the sheep imported from Australia (n=47)，respectively.However，no SBV nucleic acid-positive sample was detected.The successful development of SBV RT-qPCR provides us with an efficient detection tool for dealing with Schmallenberg disease.

CLC Number:

S852.659.5

ZHANG Yong-ning，WU Shao-qiang，LYU Ji-zhou，FENG Chun-yan，WANG Cai-xia，DENG Jun-hua，YUAN Xiang-fen，LIN Xiang-mei. A Real-time Quantitative Reverse Transcription-PCR for the Detection of Schmallenberg Virus Nucleic Acid[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(11): 1824-1829.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2014.11.012

https://www.xmsyxb.com/EN/Y2014/V45/I11/1824

References

［1］RASMUSSEN L D，KRISTENSEN B，KIRKEBY C，et al.Culicoids as vectors of Schmallenberg virus［J］.Emerg Infect Dis，2012，18(7):1204-1206.
［2］VERONESI E，HENSTOCK M，GUBBINS S，et al.Implicating Culicoides biting midges as vectors of Schmallenberg virus using semi-quantitative RT-PCR［J］.PLoS One，2013，8(3):e57747.
［3］HOFFMANN B，SCHEUCH M，HÖPER D，et al.Novel orthobunyavirus in Cattle，Europe，2011［J］.Emerg Infect Dis，2012，18(3):469-472.
［4］BILK S，SCHULZE C，FISCHER M，et al.Organ distribution of Schmallenberg virus RNA in malformed newborns［J］.Vet Microbiol，2012，159(1-2):236-238.
［5］WERNIKE K，CONRATHS F，ZANELLA G，et al.Schmallenberg virus-Two years of experiences［J］.Prev Vet Med，2014，116（4）：423-434.
［6］FISCHER M，HOFFMANN B，GOLLER K V，et al.A mutation ‘hot spot’ in the Schmallenberg virus M segment［J］.J Gen Virol，2013，94(Pt6):1161-1167.
［7］BRÉARD E，LARA E，COMTET L，et al.Validation of a commercially available indirect ELISA using a nucleocapside recombinant protein for detection of Schmallenberg virus antibodies［J］.PLoS One，2013，8(1):e53446.
［8］HOFFMANN B，SCHULZ C，BEER M.First detection of Schmallenberg virus RNA in bovine semen，Germany，2012［J］.Vet Microbiol，2013，167(3-4):289-295.
［9］ZHANG Y，WU S，WANG J，et al.Expression and purification of the nucleocapsid protein of Schmallenberg virus，and preparation and characterization of a monoclonal antibody against this protein［J］.Protein Expr Purif，2013，92(1):1-8.
［10］张永宁，吴绍强，吕继洲，等.施马伦贝格病毒核衣壳蛋白的原核表达、纯化及多克隆抗体的制备［J］.畜牧兽医学报，2013，44（10）：1629-1636.
［11］张永宁，吴绍强，WERNIKE K，等.施马伦贝格病毒核衣壳蛋白单克隆抗体的制备及其特性分析［J］.细胞与分子免疫学杂志，2014，30（3）：289-293.
［12］张永宁，吴绍强，林祥梅.施马伦贝格病研究进展［J］.畜牧兽医学报，2014，45（7）：1029-1037.
［13］DOMINGUEZ M，HENDRIKX P，ZIENTARA S，et al.Preliminary estimate of Schmallenberg virus infection impact in sheep flocks-France［J］.Vet Rec，2012，171(17):426.
［14］MARTINELLE L，DAL POZZO F，GAUTHIER B，et al.Field veterinary survey on clinical and economic impact of Schmallenberg virus in Belgium［J］.Transbound Emerg Dis，2014，61(3):285-288.
［15］BLOMSTRÖM A L，STENBERG H，SCHARIN I，et al.Serological screening suggests presence of schmallenberg virus in cattle，sheep and goat in the zambezia province，mozambique［J］.Transbound Emerg Dis，2014，61(4):289-292.
［16］HOFFMANN B，BEER M，REID S M，et al.A review of RT-PCR technologies used in veterinary virology and disease control:sensitive and specific diagnosis of five livestock diseases notifiable to the World Organisation for Animal Health［J］.Vet Microbiol，2009，139(1-2):1-23.
［17］COUPEAU D，CLAINE F，WIGGERS L，et al.In vivo and in vitro identification of a hypervariable region in Schmallenberg virus［J］.J Gen Virol，2013，94(Pt6):1168-1174.
［18］FISCHER M，SCHIRRMEIER H，WERNIKE K，et al.Development of a pan-Simbu real-time reverse transcriptase PCR for the detection of Simbu serogroup viruses and comparison with SBV diagnostic PCR systems［J］.Virol J，2013，10:327.
［19］DE REGGE N，VAN DEN BERG T，GEORGES L，et al.Diagnosis of Schmallenberg virus infection in malformed lambs and calves and first indications for virus clearance in the fetus［J］.Vet Microbiol，2013，162(2-4):595-600.

[1]	MENG Lingzhai, CHEN Chunli, YU Mengmeng, WANG Zhanxin, WANG Suyan, LIU Peng, HE Tana, GUO Ru, CHEN Yuntong, LIU Changjun, QI Xiaole, WU Zhiqiang, GAO Yulong. Pathogenicity of Avian Metapneumovirus Subtype B on Yellow Feather Broilers and Evaluation on Immune Effect of Inactivated Vaccine [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(12): 5154-5161.
[2]	JIANG Shengqiang, HU Jing, CHEN Hongying. Expression Analysis of CircRNAs in A549 Cells Infected with H1N1 Influenza A Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4724-4734.
[3]	ZHANG Ao, TAN Bin, LIU Kexin, LIU Jiali, ZHANG Shuqin. Genomic Characterization Analysis of a H1N1 Subtype Swine Influenza Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(11): 4866-4871.
[4]	HE Chenxiang, GAO Shandian, TIAN Zhancheng, DU Junzheng, WANG Jinming, GUAN Guiquan, YIN Hong. Development of a Differential Diagnostic ELISA Based on G_NS Protein to Distinguish BEFV Infected and Vaccinated Cattle [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(10): 4320-4326.
[5]	CHANG Yiming, TANG Cheng, YUE Hua. Molecular Epidemiological Investigation of Bovine Respiratory Syncytial Virus in Yaks [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2030-2041.
[6]	FANG Yuan, HOU Qiaodi, XIANG Chaohui, ZHAO Hongyi, QI Xuefeng. Regulatory Effects of IFITM3 on Proliferation of Peste des Petits Ruminants Virus (PPRV) in Goat Endometrial Epithelial Cells [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(5): 2200-2207.
[7]	LI Shun, LI Guizhen, YUAN Yaoxian, LI Kangjian, MA Chunquan, LI Shoujun, HUANG Liangzong, MA Jun. Effect of the Length of PA-X Gene on Replication and Pathogenicity of H3N2 Canine Influenza Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2023, 54(1): 272-280.
[8]	CUI Mingxian, WANG Xingbo, HUANG Yanming, BIAN Xiyi, FENG Mengke, YAN Yan, DONG Weiren, ZHOU Jiyong. Genetic Characterization and Evolution of Three Strains of H3N2 Avian Influenza Viruses [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 4116-4122.
[9]	YU Mengmeng, BAO Yuanling, WANG Suyan, XIN Ziqi, LIU Peng, FENG Xiaoyan, MENG Lingzhai, GUO Ru, ZHANG Yanping, LIU Changjun, QI Xiaole, LI Junping, WANG Xiaomei, GAO Yulong. Isolation, Identification and Pathogenicity of Subtype B Avian Metapneumovirus [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(10): 3540-3549.
[10]	ZHOU Yong, LI Zhixin, LU Hongwei, SUN Yan, LI Tian, DU Fanshu, PU Juan. Surveillance and Outbreak Analysis of H5 and H7N9 Subtypes of Highly Pathogenic Avian Influenza in China [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 3093-3106.
[11]	PAN Chunrong, DENG Ruixue, HU Linjie, ZHU Xueliang, SUN Yuefeng, WANG Guirong, MENG Xuelian. Effect of Histone Deacetylase Inhibitors on Peste Des Petits Ruminants Virus Replication [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(7): 2307-2316.
[12]	CHEN Zixuan, ZHANG Nan, HU Qun, QUAN Keji, QIN Tao, CHEN Sujuan, PENG Daxin, LIU Xiufan. Mutation Analysis of Antigen Sites 145 and 153 on Hemagglutinin of H9N2 Avian Influenza Virus in China [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1165-1172.
[13]	DUAN Zhiqiang, XIE Lingling, CHEN Jiaqi, TANG Hong, WANG Yanbi, ZHAO Caiqin, ZHAO Jiafu. Research Progress on the Interactions of Newcastle Disease Virus M Protein with Host Proteins [J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(1): 32-42.
[14]	YAN Yayao, GU Min, LIU Xiufan. Advance in the Influence of Amino Acid Variation in HA Protein on the Biological Properties of H7N9 Subtype Influenza Virus [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(8): 2093-2106.
[15]	JIANG Mei, CHEN Wu, ZHAI Junqiong, BU Wandi, XIE Yilun, LIU Canbin, SHAN Fen, LUO Manlin. Cloning and Sequence Analysis of H and F Genes of Canine Distemper Virus Strain from Red Panda [J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(6): 1670-1676.

A Real-time Quantitative Reverse Transcription-PCR for the Detection of Schmallenberg Virus Nucleic Acid

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 15

Recommended Articles

Metrics

Comments