关键词: 马铃薯, 马铃薯纺锤块茎类病毒, 双重RT-PCR, 马铃薯线粒体NADH ND2 mRNA

Abstract: 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.

Key words: Potato, Potato spindle tuber viroid, Duplex RT-PCR, Potato NADH ND2 subunit mRNA