Sensitive Detection of Viruses in Pollen Using Conventional and Real‐time Reverse Transcription‐polymerase Chain Reaction

Abstract

AbstractPollen is traded internationally as a source of germplasm and for pollination. Thirty‐nine viruses and five viroids are known to be pollen transmitted. We investigated whether reverse transcription‐polymerase chain reaction (RT‐PCR) could be used to detect viruses reliably in pollen. Four extraction methods yielded nucleic acid in appropriate quantity and quality from Tobacco ringspot virus (TRSV)‐infected pollen for RT‐PCR amplification. One method, the RNeasy® Plant Mini Kit was used subsequently to extract nucleic acid of amplifiable quality from nine plant species, and pollen infected with three ilarvirus and two nepovirus species. A real‐time TaqMan™ RT‐PCR for the detection of TRSV was reliable and specific using 167 extracts of pollen from plants of Nicotiana glutinosa. The assay was highly sensitive, with extracts testing positive to a 10−6 dilution, equivalent to a single pollen grain. This demonstrated that RT‐PCR methods can detect virus‐infected pollen reliably, sensitively and specifically. The possible application of these RT‐PCR methods to replace current quarantine procedures without compromising biosecurity is discussed.