体外转录法制备小反刍兽疫病毒荧光定量RT-PCR检测标准阳性模板  

In Vitro Transcription for Preparation of Standard Positive Template for Real -Time Quantitative Reverse Transcriptase PCR (qRT-PCR) for the Detection of Peste des Petits Ruminants Virus

作　　者：赵文姬[1,2] 包静月[1] 李林[1] 王志亮[1] 

机构地区：[1]中国动物卫生与流行病学中心国家外来动物疫病诊断中心,山东青岛266032 [2]扬州大学兽医学院,江苏扬州225009

出　　处：《中国动物检疫》2008年第12期28-30,共3页China Animal Health Inspection

摘　　要：针对小反刍兽疫病毒的F基因设计了一对荧光定量RT—PCR的引物和探针，在正向引物的5’端加上T7启动子后与反向引物扩增小反刍兽疫病毒株Nigeria75／1细胞培养液RNA．切胶回收目的条带作为体外转录模板．体外转录后测RNA浓度以及OD值。线性试验和特异性试验结果表明，该模板具有良好的线性范围和特异性。当稀释度为4．9×10^8-4．9×10^3拷贝／μL时，相关系数为0．999。该模板稳定性好。-80℃保存一个月后无显著变化。对起始浓度为4．9×10^6、4．9×10^5、4．9×10^4拷贝／μl的标准品分别检测10次，变异系数分别为1．09％，1．47％，1．34％，表明以此模板进行荧光定量PCR具有较好的重复性。Primers and probe for qRT-PCR were designed based on the fusion protein gene sequence of PPR virus. RNA extracted from the cell culture infected with PPRV vaccine strain Nigeria 75/1 was amplified by using the forward primer added with T7 promoter at the 5＂ terminal and reverse primer, and the PCR products were reclaimed as the in vitro transcription template. After in vitro transcription, OD value was detected. The template showed good linearity and specificity. The correlation coefficient is 0.999 when the template was diluted to 4.9×10^8- 4.9×10^3 copies/μL. After storing at -80℃ for one month, the template was also stable as before. And the coefficient of variation value for intra-experimental reproducibility ranged from 1.09% to 1.47%, showing an excellent repetitiveness of the template.

关 键 词：小反刍兽疫病毒 体外转录 实时定量RT—PCR 

分 类 号：S862.659.5[农业科学—野生动物驯养]