摘要
根据马铃薯纺锤块茎类病毒(PSTVd)基因序列设计特异引物,采用反转录聚合酶链式反应(RT-PCR)技术进行PSTVd检测研究。为避免由于提取的RNA降解或者RT-PCR反应质量不高所造成的假阴性问题,在检测过程中引入马铃薯线粒体NADH脱氢酶ND2亚基基因mRNA为内对照,该内对照的一个引物跨越了该基因内含子区域,只对剪接后的mRNA进行特异扩增而不扩增其本身DNA。利用内对照引物和PSTVd特异引物进行双重RT-PCR检测,分别扩增出190 bp的内对照基因特异片段和360 bp的PSTVd特异条带,与预期引物设计大小一致。引入的内对照可以较好地监控PSTVd的RT-PCR检测过程。
The specific primers were designed according to the highly conserved regions of Potato spindle tuber viroid（PSTVd）gene sequence, and were used to detect PSTVd by reverse transcription polymerase chain reaction（RT-PCR）.In order to avoid false negative results from RNA degradation or the poor reaction quality of RT-PCR, an internal control which based on the genomic mRNA sequence of potato mitochondrial NADH dehydrogenase ND2 subunit was incorporated in the PSTVd detection protocol. One of the internal control primers was designed and located over the spliced junction of mRNA, the primers were shown to amplify only the spliced RNA derived cDNA but not the genomic DNA itself. Using the internal control primers and the PSTVd primers, two specific PCR fragments were amplified by duplex RT-PCR, which were expected the size of 190 bp for internal control gene and 360 bp for PSTVd, respectively. The internal control could effectively inspect the detection process of PSTVd by duplex RT-PCR.
出处
《园艺学报》
CAS
CSCD
北大核心
2007年第5期1213-1216,共4页
Acta Horticulturae Sinica
基金
河北省科技攻关项目(04225505)
