novel pair of universal primers for the detection of potyviruses

• Published in: Plant pathology Vol. 59; no. 2; pp. 211 - 220
• Main Authors: Zheng, L, Rodoni, B.C, Gibbs, M.J, Gibbs, A.J
• Format: Journal Article
• Language: English
• Published: Oxford, UK Oxford, UK : Blackwell Publishing Ltd 01-04-2010; Blackwell Publishing Ltd; Blackwell; Wiley Subscription Services, Inc
• Subjects: Biological and medical sciences; biosecurity; degenerate primers; detection; diagnostics; Fundamental and applied biological sciences. Psychology; New species; Phytopathology. Animal pests. Plant and forest protection; plant virus; Plant viruses; Plant viruses and viroids; Polymerase chain reaction; Potyvirus; Primers; RNA; Plant pathology; biosecurity; Plant pathogen; RNA virus; Primer; Biosafety; Biological monitoring; Virus; diagnostics; Diagnosis; Potyvirus; degenerate primers; Detection; Potyviridae; Novelty; plant virus
• ISSN: 0032-0862; 1365-3059
• DOI: 10.1111/j.1365-3059.2009.02201.x

A novel pair of universal primers was developed to detect potyvirus species after conserved sites were identified using all full-length potyvirus sequences available by 2005. The breadth of specificity of the new primers, NIb2F and NIb3R, was investigated and compared with the specificity of two routinely used primer pairs in plant virus diagnostic laboratories. RNA from 40 potyvirus isolates representing 23 recognized and three possible new species was tested. Reactions with NIb2F and NIb3R produced amplicons of 350 bp from all 40 virus isolates tested. Reactions with the previously published WCIEN and Potyvirid primers amplified cDNA from 32 and 21 isolates, representing possibly 21 and 15 species, respectively. The identity of 12 unknown potyvirus isolates was confirmed by sequencing and three were found to be potentially distinct potyvirus species. Gel banding patterns from reactions with NIb2F and NIb3R were simpler to interpret than those from reactions with the other two primer sets; fewer products were visible and the cDNA fragments were less variable in size. RT-PCR with the novel primers is predicted to be able to detect virus isolates from all major groups within the genus Potyvirus and its reliability makes it well suited for use as a routine diagnostic assay.