针对猪瘟病毒E2蛋白的实时荧光定量PCR方法的建立及应用
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摘要
根据猪瘟病毒(Classical swine fever virus,CSFV)E2基因的保守序列,设计合成一套特异性引物和TaqMan探针,通过对反应体系和反应条件进行优化后,检测其特异性和灵敏性。结果显示,本研究建立的实时荧光定量PCR在101～108范围内线性相关系数为0.997,能够检测到相当于10 copies/μL的病毒核酸,比常规PCR检测方法敏感性强;采用建立的实时荧光定量PCR方法检验猪繁殖与呼吸综合征病毒、猪伪狂犬病毒和猪圆环病毒2型呈现阴性结果,特异性强;变异系数为0.09%～0.7%,小于1%,具有良好的重复性。本研究建立的猪瘟病毒实时荧光定量PCR方法为临床上猪瘟病毒的诊断和定量研究提供了重要的检测工具。
A real-time fluorescence quantitative PCR(qPCR) assay was innovated and developed for Classical swine fever virus(CSFV) for convenient, effective and rapid detection of clinical cases. A primer pairs and a probe based on the CSFV E2 gene were designed and its sensitivity and specificity were detected. The assay showed good specificity and linear relationship at a range of 101～108 with the detection limit at 10 copies/μL of viral genome. The real-time qPCR developed here was highly specific to CSFV as it did not react with Porcine reproductive and respiratory syndrome virus(PRRSV), Porcine circovirus type 2(PCV2) and Pseudorabies virus(PRV). The distributions with a coefficient of variation to be 0.09%～0.7% were less than 1%, indicating that the assay had good reproducibility. In this study, the real-time RT-qPCR assay provided a very important tool for CSFV investigation and quantitative research.
A real-time fluorescence quantitative PCR(qPCR) assay was innovated and developed for Classical swine fever virus(CSFV) for convenient, effective and rapid detection of clinical cases. A primer pairs and a probe based on the CSFV E2 gene were designed and its sensitivity and specificity were detected. The assay showed good specificity and linear relationship at a range of 101～108 with the detection limit at 10 copies/μL of viral genome. The real-time qPCR developed here was highly specific to CSFV as it did not react with Porcine reproductive and respiratory syndrome virus(PRRSV), Porcine circovirus type 2(PCV2) and Pseudorabies virus(PRV). The distributions with a coefficient of variation to be 0.09%～0.7% were less than 1%, indicating that the assay had good reproducibility. In this study, the real-time RT-qPCR assay provided a very important tool for CSFV investigation and quantitative research.
引文
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[12]Gibson U E, Heid C A, Williams P M. A novel method for real time quantitative RT-PCR[J]. Genome Res, 1996,6(10):995-1001.
[13]韦天超,田志军,安同庆,等.猪繁殖与呼吸综合征病毒Taq Man-MGB荧光定量RT-PCR方法的建立和应用[J].中国预防兽医学报, 2008, 30(12):944-948, 991.
[14]高飞,曲泽慧,姜一峰,等.重组猪瘟病毒C株E2蛋白的猪繁殖与呼吸综合征病毒的构建及鉴定[J].中国动物传染病学报, 2015, 23(5):1-9.
[15]Lin Y C, Wu S C, Yang M Y, et al. Application of realtime quantitative polymerase chain reaction to monitoring infection of classic swine fever virus and determining optimalharvesttimeinlarge-scaleproduction[J].Vaccine, 31(47):5565-5571.
[16]田青,郑浩,童武,等.伪狂犬病毒实时荧光定量PCR方法的建立[J].中国动物传染病学报, 2015, 23(3):1-6.
[2]洪健,周雪平. ICTV第八次报告的最新病毒分类系统[J].中国病毒学, 2006, 21(1):84-96.
[3]仇华吉,童光志,沈荣显.猪瘟兔化弱毒疫苗—半个世纪的回顾[J].中国农业科学,2005, 38(8):1675-1685.
[4]Tu C, Lu Z, Li H, et al. Phylogenetic comparison of classical swine fever virus in China[J]. Virus Res, 2001,81(1-2):29-37.
[5]吕宗吉,涂长春,余兴龙,等.我国猪瘟的流行病学现状分析[J].中国预防兽医学报, 2001, 23(4):300-303.
[6]Zhang H, Leng C, Feng L, et al. A new subgenotype 2.1d isolates of classical swine fever virus in China, 2014[J].Infect Genet Evol, 2015, 34:94-105.
[7]冯丽苹,张洪亮,刘春晓,等. 2015年我国部分地区2.1d新基因亚型猪瘟病毒的分子流行病学分析[J].中国预防兽医学报, 2015, 37(9):651-655.
[8]冯丽苹,张洪亮,彭金美,等.两株2.1d新基因亚型猪瘟病毒的全基因组序列分析及其基因亚型分子特征[J].中国预防兽医学报, 2016, 38(1):1-5.
[9]van Gennip H G, van Rijn P A, Widjojoatmodjo M N,et al. Chimeric classical swine fever viruses containing envelope protein E(RNS)or E2 of bovine viral diarrhea virus protect pigs against challenge with CSFV and induce a distinguishable antibody response[J]. Vaccine,2000, 19(4-5):447-459.
[10]Weiland E, Stark R, Haas B, et al. Pesitivirus glycoprotein which induces neutralizing antibodies forms part of a disulfide-linked heterodimer[J]. J Virol, 1990, 64(8):3563-3569.
[11]HeidCA,StevensJ,LivakKJ,etal.Realtime quantitative PCR methods[J]. Genome Res, 1996, 6(10):986-994.
[12]Gibson U E, Heid C A, Williams P M. A novel method for real time quantitative RT-PCR[J]. Genome Res, 1996,6(10):995-1001.
[13]韦天超,田志军,安同庆,等.猪繁殖与呼吸综合征病毒Taq Man-MGB荧光定量RT-PCR方法的建立和应用[J].中国预防兽医学报, 2008, 30(12):944-948, 991.
[14]高飞,曲泽慧,姜一峰,等.重组猪瘟病毒C株E2蛋白的猪繁殖与呼吸综合征病毒的构建及鉴定[J].中国动物传染病学报, 2015, 23(5):1-9.
[15]Lin Y C, Wu S C, Yang M Y, et al. Application of realtime quantitative polymerase chain reaction to monitoring infection of classic swine fever virus and determining optimalharvesttimeinlarge-scaleproduction[J].Vaccine, 31(47):5565-5571.
[16]田青,郑浩,童武,等.伪狂犬病毒实时荧光定量PCR方法的建立[J].中国动物传染病学报, 2015, 23(3):1-6.