A novel usage of random primers for multiplex RT-PCR detection of virus and viroid in aphids, leaves, and tubers

Abstract

A multiplex reverse transcription polymerase chain reaction (m-RT-PCR) was developed for the simultaneous detection of five potato viruses and a viroid. The synthesis of cDNAs used for amplification was primed by hexanucleotides (random primers, RP). An RNA extraction procedure employing DNase I, is routinely used to isolate potato viruses and viroid ( Potato virus S, PVS; Potato leafroll virus, PLRV; Potato virus X, PVX; Potato virus A and Y, PVA, PVY; and Potato spindle tuber viroid, PSTVd) from infected tissues. This extraction method produced deoxy-oligonucleotides, which in turn were used to prime the reverse transcription of RNA templates of all the viruses and the viroid. A time-course study from 15 s to 30 min showed optimal oligonucleotide generation by DNase I occurred at 10 min, an incubation time already incorporated in the extraction protocol. The presence of oligonucleotides capable of priming cDNA synthesis was also demonstrated in RNA preparations from aphids, leaves, and tubers. In order to duplicate the priming of templates by oligonucleotides, commercially available hexanucleotides were used as RP. When fragments were amplified from 5′- and 3′-ends of the random primed cDNA of PVY genome, bands of similar intensity were observed. In contrast, when two fragments (short and long) from the P1 gene region of the PVA genome were amplified, the yield of the short fragment was significantly higher in intensity than that of the long fragment in random primed cDNA. Irrespective of the origin of the primers (generated during extraction vs. commercially purchased), single or multiple viruses or the viroid were detected by amplification of random primed cDNAs present individually in the reaction or in a cDNA pool consisting of five viruses and the viroid. The cDNA produced by RP or virus specific primers (SP) was used to detect PLRV and PVY from infected tubers in a duplex reverse transcription polymerase chain reaction (d-RT-PCR). The RP cDNA gave increased detection. Comparison of RP primed cDNAs from dormant or sprouted tubers and leaves showed that for some cultivars, such as ‘Shepody’, leaves were more reliable for PVY and PLRV detection than the tubers, in both the d- and m-RT-PCR.