Multiplex RT-PCR detection of four aphid-borne strawberry viruses in Fragaria spp. in combination with a plant mRNA specific internal control

Abstract

The principal aphid-borne viruses infecting Strawberry ( Fragaria spp.) Strawberry crinkle virus (SCV), Strawberry mild yellow edge virus (SMYEV), Strawberry mottle virus (SMoV) and Strawberry vein banding virus (SVBV) can cause serious crop losses. In this paper, a multiplex reverse transcriptase polymerase chain reaction (RT-PCR) method is described for the simultaneous detection of all four viruses in combination with a plant mRNA specific internal control which can be used as an indicator of the effectiveness of the extraction and RT-PCR. In total, 18 strawberry isolates infected naturally were analysed by this method. Every combination of RNA virus was able to be detected and a full complement of all four viruses were found together in three isolates, all taken from wild strawberry ( Fragaria chiloensis (L.) Duch.) in Chile. The upper detection limit for the four viruses was at an extract dilution of 1/200. The broad applicability of the RNA specific internal control primers—which produced a PCR fragment of the expected size in 25 of 27 plant species tested—combined with improvements, made in extraction methods described provides potentially a standard method for comparable RT-PCR analyses in a wide variety of plant species.