Table 6. Characteristics of the oligonucleotide primers used in this study.
General assay target | Multiplex PCR primers (5′-3′)a, b | Amplicon sizec | TSPE primer sequences (5′-3′)d, e | Target | MagPlex-TAG bead addressf | ||
Pospiviroid | PospF1 | CCWGTGGTKCWCWCCTGACC | ∼270 nt | tPospUni | CTAAATCACATACTTAACAACAAA GGGATCCCCGGGGAAACCTGGA | All Pospiviroidspecies (-CLVd) | 63 |
PospF2 | GCACCCCTGACCTGCAATG | tCLVd | TTAATACAATTCTCTCTTTCTCTA AGAGCGCAAGAGCGGTCTC | CLVd | 54 | ||
PospF3 | GGTGCCTGTGGTGCCTC | tCSVd | ATACTTTACAAACAAATAACACAC AGGAAGTCCGACGAGATCGC | CSVd | 19 | ||
PospR1 | CAGTTGTWKCCACCGGGTAGT | tCEVd | ACAAATATCTAACTACTATCACAA CGGATCACTGGCGTCCAGC | CEVd | 39 | ||
PospR2 | CCACCGGTCGCGTCAG | tIrVd | CAAACAAACATTCAAATATCAATC GTCGACCGCGTAAAGACCGGA | IrVd-1 | 22 | ||
tPSTVd | TTAACAACTTATACAAACACAAAC GCGGCCGACAGGAGTAATTCC | PSTVd | 53 | ||||
tTCDVd | AATCAACACACAATAACATTCATA GCAAAAGGCGGCAGGGAGC | TCDVd | 48 | ||||
tTASVd | AATTTCTTCTCTTTCTTTCACAAT CGAGGTCGGGGGCTTCGGA | TASVd | 14 | ||||
tPCFVd | CTATCATTTATCTCTTTCTCAATT GAAAGGGGAAGCAAGCATCTCCT | PCFVd | 72 | ||||
tTPMVd | TCATCACTTTCTTTACTTTACATT GTCGCGGCTGGGGAGTCTC | TPMVde | 44 | ||||
Plant Internal control (PlantIC) | Nad5F | GATGCTTCTTGGGGCTTCTTGTT | 181 nt | tNad5 | AACTTTCTCTCTCTATTCTTATTT AGGATCCGCATAGCCCTCGATTTATGTG | Plant mRNA NADHgene | 43 |
Nad5R | CTCCAGTCACCAACATTGGCATAA |
a The nucleotides in bold are degenerate sites, with ambiguity codes as follows: W = A or T; K = G or T.
b F, R and t represent forward primer, reverse primer and target specific primer extension (TSPE) primer, respectively.
c Amplicon size differs between species of pospiviroids due to differences in length of genomes (insertions and deletions over amplification region).
d Underlined segment of primer indicates the MagPlex-TAG sequences provided by Luminex, which bind to the complementary anti-MagPlex-TAG sequenced displayed on the surface of the corresponding bead address.
e For Tomato planta macho viroid, the target of detection includes isolates of Mexican papita viroid, as this viroid is considered a conspecific.
f The MagPlex-bead product number assigned by Luminex (Luminex Corp., Austin, TX, USA).