猪乙脑病毒RT-PCR、荧光定量PCR及多重RT-PCR诊断方法建立
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摘要
猪乙型脑炎病毒(Japanese Encephalitis Virus,JEV)是二十世纪二十年代被发现并能引起母猪繁殖障碍的主要病原体之一,其特征是孕前期受到感染后,经胎盘使胚胎或胎儿受到侵袭,引起母猪流产、胚胎死亡、胎儿畸形、胎儿木乃伊化及不孕等,此外还可以引起公猪单侧或两侧性睾丸肿大,局部发热,有痛感,数天后,睾丸肿胀消退,逐渐萎缩变硬,造成精液品质不良,不能配种,同时还可以引起仔猪的皮炎水肿,其它类型的猪感染后无明显临床特征。猪乙型脑炎病毒病存在于世界各地并在大多数猪场流行,严重影响着养猪业的发展,因此本病一直是猪病研究的热点之一,JEV又常同猪伪狂犬病毒、细小病毒、圆环病毒、猪繁殖与呼吸障碍综合征病毒等混合感染而加重了其危害。近年来,JEV感染呈扩大上升的趋势,给全球养猪业带来巨大的经济损失。所以,加强对JEV的检测、监测及预防十分紧迫。
目前,猪乙型脑炎病毒病诊断方法主要有:病毒分离、血凝抑制试验和乳胶凝集试验等检测方法,其中血凝抑制试验相对简便,但由于要制备豚鼠红细胞以及被检血清必须经高岭土或其它方法处理而显得麻烦;乳胶凝集试验的灵敏度不高,尤其是对隐性感染动物往往漏检;病毒分离和鉴定结果准确可靠,但是该方法费时费力,并且需要一定的技术条件和设备;聚合酶链式反应(PCR)及荧光定量PCR诊断具有灵敏度高、特异性强、快速、简便等优点成为诊断猪乙脑病毒的研究热点。为此,我们进行了以下研究:
(一)根据已报道的JEV基因组序列,设计并合成了1对寡核苷酸引物,通过优化RT-PCR的条件,成功地从JEV感染的细胞中扩增出349 bp片段,回收PCR产物测序,回收该片段,连接到载体上,并用EcoRⅠ进行酶切,得到3015 bp、349 bp的两条带,与预期的结果相符合,证实了该扩增片段的特异性,并对PRRSV cDNA、SIV cDNA和正常PK-15细胞基因组cDNA的RT-PCR扩增均为阴性,证明了RT-PCR检测方法的特异性。RT-PCR检测JEV cDNA的最小检测量为10 fg。
(二)设计并合成引物和相对应的探针,从JEV感染的细胞中提取RNA,反转录后经PCR扩增,产物纯化后与pGEM-T-easy载体连接,转化大肠杆菌JM109,筛选后得到重组标准品质粒,对重组标准品质粒进行PCR和测序鉴定,证实目的片段已经成功克隆。将系列梯度稀释的重组标准品质粒进行荧光定量PCR,系统自动分析软件显示Ct值与标准品浓度的对数之间存在良好的线性关系。动力学曲线分析表明,在该反应体系和反应条件下,该标准曲线的灵敏度为90个拷贝。荧光PCR方法的建立,为猪乙型脑炎病毒的早期诊断、定量分析评价猪乙脑病毒感染程度奠定了基础。
(三)在建立的JEV和猪流感病毒(SIV)的单相RT-PCR基础上,通过对扩增条件的筛选,最终成功地建立了JEV和SIV的复合RT-PCR诊断方法,即利用一次RT-PCR反应,可同时扩增JEV的349 bp和SIV的155 bp特异性片段,而扩增猪圆环病毒Ⅱ型(PCV-2)及相应的培养细胞(PK-15)核酸结果均为阴性,对JEV和SIV的最低检出量分别为100 fg和10 fg的RNA。该方法适合对JEV和SIV的联合检测和鉴别诊断。
(四)在建立的JEV、猪流感病毒(SIV)和猪呼吸与繁殖障碍综合征病毒(PRRSV)的单相RT-PCR基础上,通过对扩增条件的筛选,最终成功地建立了JEV、SIV和PRRSV的复合RT-PCR诊断方法,即利用一次RT-PCR反应,可同时扩增JEV的349 bp、SIV的155 bp和PRRSV的426 bp特异性片段,而扩增猪圆环病毒Ⅱ型(PCV-2)及相应的培养细胞(PK-15)核酸结果均为阴性,对JEV、SIV和PRRSV的最低检出量分别为100 fg、10 fg和1000 fg的cDNA。该方法适合对JEV、SIV和PRRSV的联合检测和鉴别诊断。
Japanese Encephalitis Virus (JEV) causes reproductive failure in swine, manifested as embryonic resorption, fetal mummification, abortion and stillbirths. The virus is ubiquitous among swine throughout the world and is enzootic in most herds that have been tested. In this research, the RT-PCR method and fluorescence quantitative RT-PCR for the detection of JEV was developed.
1. A pair of primers was synthesized from the Japanese Encephalitis Virus cDNA sequences published in GenBank. Through optimizing RT-PCR conditions, the expected 349 bp fragment was amplified from cDNA of JEV-infected PK-15 cells, The amplified fragment was shown to be specific for JEV cDNA after digested by EcoRⅠ. This method could detect the template cDNA of 10fg at least. These results showed the RT-PCR technique is a fast, simple, economical, specific and sentitive detection method.
2. A pair of primers and the corresponding TaqMan probe were designed, the expected fragment was amplified from RNA of JEV-infected PK-15 cells, The purified PCR product was connected with pGEM-T-easy vector and then transferred into JM109.The standard recombinant plasmid was gained from positive bacterium clone.The plasmid PCR and plasmid sequence mensuration showed that the expected fragment was successfully cloned. Series of diluted standard recombinant plasmid cDNA specimen were amplified by real-time quantitative PCR, which indicate that there is a good linear function in statistics between the Ct value and the concentration gradient of standard plasmid cDNA specimen. Analysis of the dynamic curve, under this condition of the reaction, the sensitive degree is 90 copies. The construction of real-time quantitative PCR provides the basis for the early and rapid detection and analysising the infect degree of JEV .
3. On the basis of single RT-PCR for JEV and SIV established, by optimizing action conditions, The Multiplex RT-PCR was developed. The results showed that two specific fragments of 349 bp for JEV and 155 bp for SIV could be amplified in the multiple RT-PCR simultaneously. DNA fragments were not amplified from cultural cells challenged with PCV-2 and control cultural cells (PK-15), This method could detect the template cDNA of 100pg for JEV and 10pg for SIV, This method could differentiate JEV, SIV and mixed infection.
4. On the basis of single RT-PCR for JEV、SIV and PRRSV established, by optimizing action conditions, The Multiplex RT-PCR was developed. The results showed that two specific fragments of 349 bp for JEV、155 bp for SIV and 426 bp for PRRSV could be amplified in the multiple RT-PCR simultaneously. DNA fragments were not amplified from cultural cells challenged with PCV-2 and control cultural cells (PK-15), This method could detect the template cDNA of 100pg for JEV、10pg for SIV and 1000pg for PRRSV, This method could differentiate JEV, SIV, PRRSV and mixed infection.
目前,猪乙型脑炎病毒病诊断方法主要有:病毒分离、血凝抑制试验和乳胶凝集试验等检测方法,其中血凝抑制试验相对简便,但由于要制备豚鼠红细胞以及被检血清必须经高岭土或其它方法处理而显得麻烦;乳胶凝集试验的灵敏度不高,尤其是对隐性感染动物往往漏检;病毒分离和鉴定结果准确可靠,但是该方法费时费力,并且需要一定的技术条件和设备;聚合酶链式反应(PCR)及荧光定量PCR诊断具有灵敏度高、特异性强、快速、简便等优点成为诊断猪乙脑病毒的研究热点。为此,我们进行了以下研究:
(一)根据已报道的JEV基因组序列,设计并合成了1对寡核苷酸引物,通过优化RT-PCR的条件,成功地从JEV感染的细胞中扩增出349 bp片段,回收PCR产物测序,回收该片段,连接到载体上,并用EcoRⅠ进行酶切,得到3015 bp、349 bp的两条带,与预期的结果相符合,证实了该扩增片段的特异性,并对PRRSV cDNA、SIV cDNA和正常PK-15细胞基因组cDNA的RT-PCR扩增均为阴性,证明了RT-PCR检测方法的特异性。RT-PCR检测JEV cDNA的最小检测量为10 fg。
(二)设计并合成引物和相对应的探针,从JEV感染的细胞中提取RNA,反转录后经PCR扩增,产物纯化后与pGEM-T-easy载体连接,转化大肠杆菌JM109,筛选后得到重组标准品质粒,对重组标准品质粒进行PCR和测序鉴定,证实目的片段已经成功克隆。将系列梯度稀释的重组标准品质粒进行荧光定量PCR,系统自动分析软件显示Ct值与标准品浓度的对数之间存在良好的线性关系。动力学曲线分析表明,在该反应体系和反应条件下,该标准曲线的灵敏度为90个拷贝。荧光PCR方法的建立,为猪乙型脑炎病毒的早期诊断、定量分析评价猪乙脑病毒感染程度奠定了基础。
(三)在建立的JEV和猪流感病毒(SIV)的单相RT-PCR基础上,通过对扩增条件的筛选,最终成功地建立了JEV和SIV的复合RT-PCR诊断方法,即利用一次RT-PCR反应,可同时扩增JEV的349 bp和SIV的155 bp特异性片段,而扩增猪圆环病毒Ⅱ型(PCV-2)及相应的培养细胞(PK-15)核酸结果均为阴性,对JEV和SIV的最低检出量分别为100 fg和10 fg的RNA。该方法适合对JEV和SIV的联合检测和鉴别诊断。
(四)在建立的JEV、猪流感病毒(SIV)和猪呼吸与繁殖障碍综合征病毒(PRRSV)的单相RT-PCR基础上,通过对扩增条件的筛选,最终成功地建立了JEV、SIV和PRRSV的复合RT-PCR诊断方法,即利用一次RT-PCR反应,可同时扩增JEV的349 bp、SIV的155 bp和PRRSV的426 bp特异性片段,而扩增猪圆环病毒Ⅱ型(PCV-2)及相应的培养细胞(PK-15)核酸结果均为阴性,对JEV、SIV和PRRSV的最低检出量分别为100 fg、10 fg和1000 fg的cDNA。该方法适合对JEV、SIV和PRRSV的联合检测和鉴别诊断。
Japanese Encephalitis Virus (JEV) causes reproductive failure in swine, manifested as embryonic resorption, fetal mummification, abortion and stillbirths. The virus is ubiquitous among swine throughout the world and is enzootic in most herds that have been tested. In this research, the RT-PCR method and fluorescence quantitative RT-PCR for the detection of JEV was developed.
1. A pair of primers was synthesized from the Japanese Encephalitis Virus cDNA sequences published in GenBank. Through optimizing RT-PCR conditions, the expected 349 bp fragment was amplified from cDNA of JEV-infected PK-15 cells, The amplified fragment was shown to be specific for JEV cDNA after digested by EcoRⅠ. This method could detect the template cDNA of 10fg at least. These results showed the RT-PCR technique is a fast, simple, economical, specific and sentitive detection method.
2. A pair of primers and the corresponding TaqMan probe were designed, the expected fragment was amplified from RNA of JEV-infected PK-15 cells, The purified PCR product was connected with pGEM-T-easy vector and then transferred into JM109.The standard recombinant plasmid was gained from positive bacterium clone.The plasmid PCR and plasmid sequence mensuration showed that the expected fragment was successfully cloned. Series of diluted standard recombinant plasmid cDNA specimen were amplified by real-time quantitative PCR, which indicate that there is a good linear function in statistics between the Ct value and the concentration gradient of standard plasmid cDNA specimen. Analysis of the dynamic curve, under this condition of the reaction, the sensitive degree is 90 copies. The construction of real-time quantitative PCR provides the basis for the early and rapid detection and analysising the infect degree of JEV .
3. On the basis of single RT-PCR for JEV and SIV established, by optimizing action conditions, The Multiplex RT-PCR was developed. The results showed that two specific fragments of 349 bp for JEV and 155 bp for SIV could be amplified in the multiple RT-PCR simultaneously. DNA fragments were not amplified from cultural cells challenged with PCV-2 and control cultural cells (PK-15), This method could detect the template cDNA of 100pg for JEV and 10pg for SIV, This method could differentiate JEV, SIV and mixed infection.
4. On the basis of single RT-PCR for JEV、SIV and PRRSV established, by optimizing action conditions, The Multiplex RT-PCR was developed. The results showed that two specific fragments of 349 bp for JEV、155 bp for SIV and 426 bp for PRRSV could be amplified in the multiple RT-PCR simultaneously. DNA fragments were not amplified from cultural cells challenged with PCV-2 and control cultural cells (PK-15), This method could detect the template cDNA of 100pg for JEV、10pg for SIV and 1000pg for PRRSV, This method could differentiate JEV, SIV, PRRSV and mixed infection.
引文
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