. 2014 Jan 3;9(1):e84743. doi: 10.1371/journal.pone.0084743

Table 6. Characteristics of the oligonucleotide primers used in this study.

General assay target	Multiplex PCR primers (5′-3′)^{a, b}		Amplicon size^c	TSPE primer sequences (5′-3′)^{d, e}		Target	MagPlex-TAG bead address^f
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	TPMVd^e	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).