Cytochrome c oxidase mRNA as an internal control for detection of Potato virus Y and Potato leafroll virus from single aphids by a co-amplification RT-PCR assay.

Using cytochrome c oxidase subunit 1 (COX1) mRNA as the internal control, a triplex reverse transcription-polymerase chain reaction (RT-PCR) for detection of Potato virus Y (PVY) and Potato leafroll virus (PLRV) with co-amplification of COX1 from single specimens of various aphid species has been developed. Partial length cDNA of COX1 from green peach aphid, Myzus persicae (Sulzer), potato aphid, Macrosiphum euphorbiae (Thomas), buckthorn aphid, Aphis nasturtii (Kaltenbach), and pea aphid, Acyrthosiphom pisum (Harris), was cloned and sequenced. These sequences, together with existing COX1 sequences from other aphid species capable or suspected to be capable of transmitting PVY and/or PLRV, were analyzed. The sequence identity between any two aphid species ranged from 97 to 100% at the putative protein level, and 89 to 94% at the nucleic acid level. Two highly conserved COX1 nucleotide sequence stretches were selected to design universal primers Aph F and Aph R. This primer pair, together with two existing universal primer pairs (C1-J-2183 and C1-N-2329; Favret F and Favret R), were evaluated at the optimal annealing temperature using RNA from M. persicase, M. euphorbiae, and A. nasturtii. The Aph primer pair performed well in the monoplex RT-PCR but poorly in the triplex RT-PCR in the presence of the PVY- and PLRV-specific primers. On the other hand, the Favret and C1 primer pairs performed well in both monoplex and triplex RT-PCR formats using single aphids of M. persicase, M. euphorbiae and A. nasturtii, demonstrating their suitability to indicate the successfulness of RT-PCR assays for PVY and PLRV. Using the Favret, PVY and PLRV primer sets, single aphids of M. persicase, M. euphorbiae and A. nasturtii that had been exposed to PLRV-infected and/or PVY-infected potato plants were assessed for their acquisition of the viruses by the triplex RT-PCR assay. Although majority (175/180) of the aphid samples produced the COX1 fragment, five aphid samples failed to produce either the COX1- or the virus-specific band, indicating failed RT-PCR in these samples. This method offers a sensitive tool for detection of viruliferous aphids combined to an effective quality control measure.