猪伪狂犬病病毒SYBR GreenⅠ实时荧光定量PCR方法的建立

doi:10.16431/j.cnki.1671-7236.2016.07.009

《中国畜牧兽医》---唯一指定的官方网站 ›› 2016, Vol. 43 ›› Issue (7): 1717-1722.doi: 10.16431/j.cnki.1671-7236.2016.07.009

猪伪狂犬病病毒SYBR GreenⅠ实时荧光定量PCR方法的建立

陈如敬^1,2, 吴学敏^1,2, 陈秋勇³, 严山^1,2, 车勇良^1,2, 王晨燕^1,2, 王隆柏^1,2, 周伦江^1,2

1. 福建省农业科学院畜牧兽医研究所, 福州 350013;
2. 福建省畜禽疫病防治工程中心, 福州 350013;
3. 福建农林大学动物科学学院, 福州 350002

收稿日期:2016-01-06 出版日期:2016-07-20 发布日期:2016-07-22
通讯作者: 周伦江 E-mail:lunjiang@163.com
作者简介:陈如敬(1984-),男,福建福州人,硕士,助理研究员,研究方向:动物传染病,E-mail:fjchenrujing@163.com
基金资助:
福建省属公益类科研专项（2014R1023-13、2015R1023-10）

Establishment of SYBR GreenⅠReal-time Quantitative PCR for Detection Porcine Pseudorabies Virus

CHEN Ru-jing^1,2, WU Xue-min^1,2, CHEN Qiu-yong³, YAN Shan^1,2, CHE Yong-liang^1,2, WANG Chen-yan^1,2, WANG Long-bai^1,2, ZHOU Lun-jiang^1,2

1. Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou 350013, China;
2. Fujian Animal Disease Control Technology Development Center, Fuzhou 350013, China;
3. College of Animal Sciences, Fujian Agricultural and Forestry University, Fuzhou 350002, China

Received:2016-01-06 Online:2016-07-20 Published:2016-07-22

摘要/Abstract

摘要：

本研究根据猪伪狂犬病病毒（pseudorabies virus，PRV）的gE基因序列特点，设计特异性实时荧光定量引物，建立基于SYBR GreenⅠ染料的猪PRV实时荧光定量PCR方法。结果表明，建立的实时荧光定量PCR方法检测猪PRV gE基因在7.53×10¹~7.53×10⁶ 拷贝/μL范围内有较好的线性关系；对猪圆环病毒（porcine circovirus 2，PCV2）、猪细小病毒（porcine parvovirus，PPV）、副猪嗜血杆菌（Haemophilus parasuis）和猪链球菌（Streptococcus susi）核酸均未见阳性扩增信号；从生成的熔解曲线可见，建立的方法仅对猪PRV检测出现单一特异峰，其熔解温度（Tm值）为（92.9±0.1）℃，PCV2、PPV、副猪嗜血杆菌和猪链球菌均未见特异性峰值；建立的实时荧光定量PCR方法的组内和组间变异系数分别为0.31%~1.14%和0.42%~1.74%。以上结果表明，本研究建立的猪PRV实时荧光定量PCR方法敏感性高、特异性强、重复性好，为深入开展猪PRV对宿主致病机制的研究提供检测手段。

关键词: 猪伪狂犬病毒; gE基因; SYBR GreenⅠ; 实时荧光定量PCR

Abstract:

In this study,a SYBR GreenⅠ dye based on Real-time quantitative PCR was established using the specific primers-pair according to gE gene characterization of porcine pseudorabies virus(PRV).The optimized results demonstrated that the detection assay with good linear determination range from 7.53×10¹ to 7.53×10⁶ copies per reaction.There was no cross-reaction occurred with nucleic acids extracted from the common porcine infectious diseases,such as porcine circovirus 2 (PCV2),porcine parvovirus (PPV),Haemophilus parasuis and Streptococcus susi,no amplification signals were detected.The results showed that the melting curve analysis with only one specific peaked with a melting temperature (Tm),which was (92.9±0.1)℃ at detecting PRV positive samples,also no specific peak could be detected for common porcine infectious diseases described above.Series experiments were carried out in order to assess the sensitivity,specificity and reproducibility for the method,following by the intra-assay and inter-assay CVs for Ct values obtained with the standard plasmids.The intra-assay and inter-assay statistics were 0.31% to 1.14% and 0.42% to 1.74%,respectively.All the results showed that the established method was sensitive,specific and reproducible,which meaned it could be used for the research of pathogenic mechanism of porcine pseudorabies virus.

Key words: porcine pseudorabies virus; gE gene; SYBR Green Ⅰ; Real-time quantitative PCR

中图分类号:

S852.65⁺5

陈如敬, 吴学敏, 陈秋勇, 严山, 车勇良, 王晨燕, 王隆柏, 周伦江. 猪伪狂犬病病毒SYBR GreenⅠ实时荧光定量PCR方法的建立[J]. 《中国畜牧兽医》---唯一指定的官方网站, 2016, 43(7): 1717-1722.

CHEN Ru-jing, WU Xue-min, CHEN Qiu-yong, YAN Shan, CHE Yong-liang, WANG Chen-yan, WANG Long-bai, ZHOU Lun-jiang. Establishment of SYBR GreenⅠReal-time Quantitative PCR for Detection Porcine Pseudorabies Virus[J]. , 2016, 43(7): 1717-1722.

参考文献

[1] Klupp B,Hengartner C,Mettenleiter T,et al.Complete,annotated sequence of the pseudorabies virus genome[J].J Virol,2004,78(1):424-440.
[2] Szpara M,Tafuri Y,Parsons L,et al.A wide extent of inter-strain diversity in virulent and vaccine strains of alphaherpesviruses[J].PLoS Pathog,2011,7(10):E1002282.
[3] 张青占.变异猪伪狂犬病毒的分离鉴定及生物学特性分析[D].北京:中国农业科学院,2013.
[4] Ye C,Zhang Q,Tian Z,et al.Genomic characterization of emergent pseudorabies virus in China reveals marked sequence divergence:Evidence for the existence of two major genotypes[J]. Virology,2015,483:32-43.
[5] Fuchs W,Klupp B,Granzow H,et al.Physical interaction between envelope glycoproteins E and M of pseudorabies virus and the major tegument protein UL49[J].J Virol,2002,76:8208-8217.
[6] 马力,杨丽梅,徐倩倩,等.猪伪狂犬病病毒gE蛋白在野毒诊断中的应用进展[J].中国畜牧兽医,2014,41(2):249-253.
[7] Duale H,Lyttle T,Smith B,et al.Noxious colorectal distention in spinalized rats reduces pseudorabies virus labeling of sympathetic neurons[J].J Neurotrauma,20010,27(8):1369-1378.
[8] 李碧,朱玲,周远成,等.伪狂犬病毒神经传导研究[J].病毒学报,2014,30(3):332-337.
[9] 吴学敏,陈如敬,车勇良,等.猪伪狂犬病病毒与猪圆环病毒2型双重PCR检测方法的建立及初步应用[J].中国人兽共患病学报,2015,31(7):631-634.
[10] 车勇良,俞伏松,陈少莺,等.PRV FB弱毒株与FA株gE、gI基因的同源性研究[J].西北农林科技大学学报(自然科学版),2006,34:37-41.
[11] 车勇良,陈如敬,江斌,等.副猪嗜血杆菌oppA基因的克隆、表达及间接ELISA检测方法的建立[J].福建农林大学学报(自然科学版),2015,44(3):282-288.
[12] 刘玉涛,吴秋玉,王隆柏,等.一株猪链球菌的分离与鉴定[J].福建畜牧兽医,2014,5:20-21.
[13] 刘志杰,任慧英,温建新,等.用地高辛标记的核酸探针检测猪伪狂犬病毒野毒感染的研究[J].畜牧兽医学报,2008,39(11):1621-1624.
[14] 刘园园,吴晖,肖性龙,等.猪伪狂犬病病毒TaqMan-MGB荧光定量PCR检测方法的建立及应用[J].中国畜牧兽医,2009,36(4):76-79.
[15] An T,Peng J,Tian Z,et al.Pseudorabies virus variant in Bartha-K61-vaccinated pigs,China,2012[J].Emerg Infect Dis,2013,19(11):1749-1755.
[16] 彭金美,安同庆,赵鸿远,等.猪伪狂犬病病毒新流行株的分离鉴定及抗原差异性分析[J].中国预防兽医学报,2013,35(1):1-4.
[17] Luo Y,Li N,Cong X,et al.Pathogenicity and genomic characterization of a pseudorabies virus variant isolated from Bartha-K61-vaccinated swine population in China[J].Vet Microbiol,2014,174 (1-2):107-115.
[18] Tong W,Liu F,Zheng H,et al.Emergence of a pseudorabies virus variant with increased virulence to piglets[J].Vet Microbiol,2015,181(3-4):236-240.
[19] Wang T,Xiao Y,Yang Q,et al.Construction of a gE-deleted pseudorabies virus and its efficacy to the new-emerging variant PRV challenge in the form of killed vaccine[J]. Biomed Res Int,2015,2015:684945.
[20] Gu Z,Dong J,Wang J,et al.A novel inactivated gE/gI deleted pseudorabies virus (PRV) vaccine completely protects pigs from an emerged variant PRV challenge[J].Virus Res,2015,195:57-63.
[21] Hu R,Zhou Q,Song W,et al.Novel pseudorabies virus variant with defects in TK,gE and gI protects growing pigs against lethal challenge[J]. Vaccine,2015,33(43):5733-5740.
[22] Lerma L,Munoz A,Wagner S,et al.Construction of recombinant pseudorabies viruses by using PRV BACs deficient in IE180 or pac sequences:Application of vBAC90D recombinant virus to production of PRV amplicons[J]. Virus Res, 2016,213:274-282.
[23] Liang X,Sun L,Yu T,et al.A CRISPR/Cas9 and Cre/Lox system-based express vaccine development strategy against re-emerging pseudorabies virus[J].Sci Rep, 2016,6:19176.
[24] Sun Y,Luo Y,Wang C,et al.Control of swine pseudorabies in China:Opportunities and limitations[J].Vet Microbiol,2016,183:119-124.

猪伪狂犬病病毒SYBR GreenⅠ实时荧光定量PCR方法的建立

Establishment of SYBR GreenⅠReal-time Quantitative PCR for Detection Porcine Pseudorabies Virus

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

本文评价

[1]	麻宝艺, 盛陈艳, 刘宏莹, 马青霞, 汪婷婷, 李健明, 时坤, 杜锐, 冷雪. BVDV 1型和2型TaqMan双重实时荧光定量PCR检测方法的建立[J]. 中国畜牧兽医, 2022, 49(6): 2326-2335.
[2]	吕晓楠, 徐立蒲, 张文, 王娜, 曹欢, 王小亮, 王姝, 王静波. 感染鲤浮肿病毒镜鲤的组织病理变化及病毒分布规律研究[J]. 中国畜牧兽医, 2022, 49(3): 1077-1084.
[3]	张军芳, 孙建富, 孙斌, 唐琳, 王英, 王恩泽, 崔岩, 李强, 严昌国, 李香子. 延边牛PPARγ基因的克隆及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(8): 2695-2704.
[4]	苗曼宁, 郭益文, 胡德宝, 曾雨晗, 李新, 张林林, 丁向彬, 郭宏. MSTN基因编辑牛肌肉转录组测序及生物信息学分析[J]. 中国畜牧兽医, 2021, 48(7): 2271-2281.
[5]	徐宁, 葛桂阳, 李东丽, 刘艺, 宫庆龙, 麻宝艺, 盛陈艳, 时坤, 李健明, 冷雪, 杜锐. 猪伪狂犬病毒的分离鉴定及其部分毒力基因序列遗传变异分析[J]. 中国畜牧兽医, 2021, 48(5): 1794-1803.
[6]	李恒, 字向东. 牦牛跨膜附睾蛋白1基因的克隆及其在卵丘卵母细胞复合体中的表达分析[J]. 中国畜牧兽医, 2021, 48(4): 1284-1293.
[7]	李建梅, 季正剑, 刘梅, 沈欣悦, 程旭, 俞燕, 戴亚斌. 藏鸡和隐性白羽鸡感染柔嫩艾美耳球虫后免疫相关基因的表达变化分析[J]. 中国畜牧兽医, 2021, 48(4): 1440-1448.
[8]	陈磊, 袁枫, 贺三刚, 玛依拉, 李文蓉. 绵羊FGF10基因克隆、序列分析及其在毛囊发育中的表达分析[J]. 中国畜牧兽医, 2021, 48(2): 425-432.
[9]	戴政列, 朱静静, 陈振宇, 周晓龙, 汪涵, 李向臣, 赵阿勇, 杨松柏. 猪SENP1基因克隆、生物信息学分析与表达研究[J]. 中国畜牧兽医, 2021, 48(12): 4382-4390.
[10]	姜懿轩, 王亚玲, 邢珊珊, 宋国华, 许会芬, 李明. 新西兰白兔MSTN基因克隆、生物信息学分析及表达谱构建[J]. 中国畜牧兽医, 2021, 48(10): 3501-3511.
[11]	李志强, 陶晨雨, 魏奥龙, 贾青. 猪性控精子纯度实时荧光定量PCR检测方法的建立[J]. 中国畜牧兽医, 2021, 48(10): 3682-3690.
[12]	王征帆, 朱利塞, 王娟, 李祎云, 向蕊, 应碧云, 王贵平, 贾爱卿, 白挨泉. PDCoV与TGEV双重实时荧光定量PCR检测方法的建立及初步应用[J]. 中国畜牧兽医, 2021, 48(10): 3855-3863.
[13]	李安奇, 蒋蕾, 苏晓彤, 昝林森, 王洪宝. 秦川牛ACTC1基因的表达特性分析[J]. 中国畜牧兽医, 2020, 47(8): 2359-2367.
[14]	李京徽, 邢思远, 王希彩, 李庆贺, 赵桂苹, 张永宏, 文杰, 刘冉冉. 鸡肌内脂肪沉积相关候选基因筛选[J]. 中国畜牧兽医, 2020, 47(6): 1828-1836.
[15]	姜徽, 刘攀, 李媛媛, 刘奎国, 黄锡霞, 刘江卫, 胥磊, 杜建文, 桑扎根, 杨雪梅, 赵番番, 王丹. 新疆褐牛乳房炎抗性相关基因的表达及生物信息学分析[J]. 中国畜牧兽医, 2020, 47(1): 90-97.