猪PEDV与PDCoV二重RT-PCR检测方法的建立

doi:10.13560/j.cnki.biotech.bull.1985.2018-0365

生物技术通报 ›› 2018, Vol. 34 ›› Issue (11): 205-209.doi: 10.13560/j.cnki.biotech.bull.1985.2018-0365

猪PEDV与PDC_oV二重RT-PCR检测方法的建立

孔维欢^1,2, 王晶晶^1,2, 万鹏程², 石国庆²

1. 石河子大学动物科技学院,石河子 832000;
2. 新疆农垦科学院畜牧兽医研究所,石河子 832000

收稿日期:2018-04-17 出版日期:2018-11-26 发布日期:2018-11-28
作者简介:孔维欢,男,硕士研究生,研究方向：动物遗传育种与繁殖;E-mail：919282632@qq.com
基金资助:
国家重点研发计划项目（2017YFD0501904,2017-2020）

Establishment of a Duplex RT-PCR Detection Method for Porcine PEDV and PDC_OV

KONG Wei-huan^1,2, Wang Jing-jing^1,2, WAN Peng-cheng², SHI Guo-qing²

1. College of Animal Science and Technology,Shihezi University,Shihezi 832000;
2. Animal Husbandry and Veterinary Institute,Xinjiang Academy of Agricultural and Reclamation Science,Shihezi 832000

Received:2018-04-17 Published:2018-11-26 Online:2018-11-28

摘要/Abstract

摘要： 旨在建立能同时检测出猪流行性腹泻病毒（Porcine epidemic diarrhea virus,PEDV）和猪丁型冠状病毒（Porcine delta corona virus,PDC_OV）的二重RT-PCR检测方法。根据GenBank已收录发表的PEDV和PDC_OV基因序列设计2对特异性引物,首先运用RT-PCR反应技术,通过PEDV和PDC_OV病毒的目的基因的单项扩增,对反应条件进行优化。然后通过特异性试验、敏感性试验以及临床样品的检测验证确定二重RT-PCR方法的建立。结果显示,目的基因PEDV-M扩增的片段大小是750 bp,PDC_OV-N扩增的片段大小是372 bp;能够同时检测出PEDV和PDC_OV目的基因,却检测不到PRV、CSFV、PPV三种病毒;体系优化扩增PEDV-PDC_OV混合核酸浓度下限为100 pg/μL;能够初步诊断出临床疑似病毒感染的病例。结果表明,成功的建立PEDV-PDC_OV二重RT-PCR检测方法,此方法敏感性高、特异性强,是一种能够快速有效的检测出猪PEDV-PDC_OV单病毒感染或多病毒混合感染的临床诊断方法。

关键词: PEDV, PDC_OV, RT-PCR, 临床诊断

Abstract: The aim of this study is to establish a double RT-PCR assay for simultaneously detecting porcine epidemic diarrhea virus（PEDV）and porcine delta corona virus（PDC_OV）. Two pairs of specific primers were designed according to the sequences of PEDV and PDC_OV genes published in GenBank. Firstly,the RT-PCR reaction technique was used to optimize the reaction conditions through the single amplification of the target genes of PEDV and PDC_OV viruses. Then,the establishment of double RT-PCR method was confirmed by specificity test,sensitivity test and clinical sample detection. Results showed as：The size of the amplified target gene PEDV-M was 750 bp,and 372 bp for PDC_OV-N fragment;PEDV and PDC_OV genes were detected simultaneously,but 3 viruses of PRV,CSFV and PPV were not detected. The minimum concentration limit of mixed nucleic acid concentration for optimizing and amplifying PEDV-PDC_OV was 100 pg/μL,thus it can be used to identify the clinical suspected virus infection. The results showed that a double RT-PCR detection method for PEDV-PDC_OV virus is established successfully,this method is highly sensitive and specific and a rapid and effective method for the clinical detection of PEDV-PDC_OV single virus infection or multi-virus mixed infection in pigs.

Key words: PEDV, PDC_OV, RT-PCR, clinical diagnosis

孔维欢, 王晶晶, 万鹏程, 石国庆. 猪PEDV与PDC_oV二重RT-PCR检测方法的建立[J]. 生物技术通报, 2018, 34(11): 205-209.

KONG Wei-huan, Wang Jing-jing, WAN Peng-cheng, SHI Guo-qing. Establishment of a Duplex RT-PCR Detection Method for Porcine PEDV and PDC_OV[J]. Biotechnology Bulletin, 2018, 34(11): 205-209.

参考文献

[1] Wang L, Byrum B, et al.New variat of porcie epidemic diarrhea virus, United States[J]. Emerg Infec Dis, 2014, 20(5):917.
[2] Ye C, Zhang QZ, Tian ZJ, et al.Genomic characterization of emergent pseudorabies virus in China reveals marked sequence divergence:Evidence for the existence of two major genotypes[J]. Virology, 2015, 483:473-474.
[3] Chen F, Zhu Y, Wu M, et al.Comparative genomic analysis of classical and variant virulent parental/attenuated strains of porcine epidemic diarrhea virus[J]. Viruses, 2015, 7:5525-5538.
[4] Sun RQ, Cai RJ, Chen YQ, et a1. Outbreak of porcine epidemic diarrhea in suckling piglets, China[J]. Emerg Infect Dis, 2012, 18(1):161-163.
[5] Wang L, Byrum B, Zhang Y.Detection and genetic characterization of deltacoronavirus in pigs, USA,[J]. Emerg Infect Dis, 2014, 20(7):1227-1230.
[6] Woo PC, Lau SK, et al.Discovery of seven novel mammalian and avian coronaviruses in the genus delta coronavirus supports bat coro-naviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus[J]. J Virol, 2012, 86(7):3995-4008.
[7] Ma Y, Zhang Y, et al.Origin evolution and virulence of porcine deltacoronaviruses in the United States[J]. MBio, 2015, 6(2):64.
[8] Lee S, Lee C.Complete genome characterization of Korean porcine Deltacoronavirus strain KOR/KNU14-04/2014[J]. Genome Ann-ounc, 2014, 2(6):1-2.
[9] Dong N, Fang L, Zeng S, et al.Porcine deltacoronavirus in mainland China[J]. Emerg Infect Dis, 2015, 21(12):2254-2255.
[10] Hu H, Jung K, Vlasova AN, et al.Isolation and characteri-zation of porcine deltacoronavirus from pigs with diarrhea in the United States[J]. J Clin Microbiol, 2015, 53(5):1537-1548.
[11] Jung K, Hu H, Eyerly B, et al.Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs[J]. Emerg Infect Dis, 2015, 21(4):650-654.
[12] 陈焕春. 当前中国猪病流行动态与防治[J]. 对策兽医导刊, 2011, 11:12-14.
[13] 王隆柏, 张志刚, 苏永裕, 等. 5种猪病多重PCR检测方法的建立[J]. 中国畜牧兽医, 2014, 41(2):21-25.
[14] 姜永厚, 徐辉, 商晗武, 等. 多重PCR同时检测猪圆环病毒2型、猪细小病毒、猪繁殖与呼吸综合征病毒和猪瘟病毒[J]. 中国兽医学报, 2009, 29(10):1237-1241.
[15] 王娟萍, 姚敬明, 赵喜有, 等. 猪细小病毒、猪伪狂犬病病毒和猪圆环病毒2型多重PCR检测方法的建立与应用[J]. 中国畜牧兽医, 2012, 39(8):45-52.

猪PEDV与PDC_oV二重RT-PCR检测方法的建立

Establishment of a Duplex RT-PCR Detection Method for Porcine PEDV and PDC_OV

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猪PEDV与PDCoV二重RT-PCR检测方法的建立

Establishment of a Duplex RT-PCR Detection Method for Porcine PEDV and PDCOV

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猪PEDV与PDC_oV二重RT-PCR检测方法的建立

Establishment of a Duplex RT-PCR Detection Method for Porcine PEDV and PDC_OV