Development of universal primers for detection of potato carlaviruses by RT-PCR.
To facilitate efficient and accurate detection of potato-infecting carlaviruses, degenerated universal primers were designed based on conserved amino acid and nucleotide sequences. Two sense primers, Car-F1 and Car-F2, were based on the amino acid sequences "SNNMA" and "GLGVPTE", respectively, in the coat protein. The reverse primer, Car-R, which was located at the border of the nucleic acid binding protein gene and the 3' untranslated region, and dT-B, which was derived from the oligo-dT targeting the poly(A) tail, were selected. Successful application of fragments within the predicted size range of carlaviruses was obtained using Car-F1 paired with either Car-R or dT-B from tested carlaviruses (Potato virus S, M and latent) by RT-PCR. The Car-F2 failed to yield clear-cut fragments within the predicted size range when paired with either Car-R or dT-B in RT-PCR. However, a less degenerated version of the primer, Car-F2b, resulted in amplicons within the predicted size range when paired with either Car-R or dT-B. Sequencing of the tentative carlavirus-fragments resulting from Car-F1/Car-R and Car-F2b/dT-B proved their carlavirus-origin, thus indicating the high specificity of these primers. The sensitivity of Car-F1/Car-R or Car-F2b/Car-R mediated RT-PCR for the detection of carlavirus-infected potato tubers were assessed using composite samples containing one carlavirus-infected-potato-tuber RNA sample with up to 49 virus-free-potato-tuber RNA samples under the optimal annealing temperature. The target carlaviruses were detected readily from all composites, demonstrating a high sensitivity. The method was further evaluated using presumed virus-free or carlavirus-infected potatoes of several cultivars, and reliable results were obtained.
Nie
Xianzhou
X
Potato Research Centre, Agriculture and Agri-Food Canada, P.O. Box 20280, 850 Lincoln Road, Fredericton, New Brunswick E3B 4Z7, Canada. niex@agr.gc.ca
Bai
Yanju
Y
Molen
Teresa A
TA
Desjardins
David C
DC
D020413:3' Untranslated Regions / Q000235:genetics; D036022:Capsid Proteins / Q000235:genetics; D017788:Carlavirus / Q000235:genetics / Q000302:isolation & purification*; D017931:DNA Primers / Q000235:genetics; D008969:Molecular Sequence Data; D010935:Plant Diseases / Q000821:virology*; D012367:RNA, Viral / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000821:virology*
D020413:3' Untranslated Regions; D036022:Capsid Proteins; D017931:DNA Primers; D012367:RNA, Viral