摘要
选取猪伪狂犬病毒(PRV)基因组中gE基因,设计特异性引物和Taqm an探针,利用实时定量PCR来定量检测PRV。利用PCR技术扩增猪伪狂犬病毒gE基因80 bp片段,并克隆到pMD18-T载体上,阳性重组质粒命名为pgE80。pgE80质粒进行10倍系列稀释,作为荧光PCR检测的标准模板,定量拷贝数,并绘制标准曲线。以提取的PRV、猪圆环病毒2型(PCV2)、猪细小病毒(PPV)、猪瘟病毒(CSFV)、猪繁殖与呼吸障碍综合症病毒(PRRSV)和传染性胃肠炎病毒(TGEV)的DNA作为模板,进行特异性检测。该实时定量PCR方法,标准曲线斜率为-0.37,R2=0.992,可以检测到20个拷贝数。PRV能被扩增出S型荧光曲线,而其它病毒均未能扩出。用PRV强毒肌肉注射仔猪,定量PCR比普通PCR更能灵敏而特异地检出呼吸道排毒和血液带毒。建立的实时定量PCR方法可用于临床样品快捷、准确、方便地检测。
A quantitative real-time PCR assay was established for detection of pseudorabies virus. The primers and probe were designed according to the gE gene sequence of pseudorabies virus. The 80 bp region of the PRV gE gene was cloned into pMD18-T vector, and the constructed plasmid was named pgE80. Serial dilutions of plasmid pgE80 were used to quantify the virus genomic copy number, and served as standard curve. PRV, PCV2, PPV, CSFV, PRRSV and TGEV DNA （cDNA） were detected at the same time with the real time qPCR. Results showed that the real time qPCR was sensitive and specific. Regression coefficient of the quantitative curve was 0. 992. Twenty copies of DNA could be detected and S curve can be amplified only from PRV genomic DNA, not from other viruses. After intramuscular injection of high virulent PRV, the virus in blood and nasal secretion can be detected more sensitively by qRT-PCR than by the traditional PCR.
出处
《江苏农业学报》
CSCD
北大核心
2008年第4期440-443,共4页
Jiangsu Journal of Agricultural Sciences
基金
国家"973"计划前期专项(2007CB116308)
