TABLE 1.

Quantitative real-time PCR primer and probe sets for the detection of reverse transcription intermediates

RT step detecteda	Oligonucleotide nameb	Sequencec	Binding positiond
Minus-strand strong stop	OJWB45	GCGCCAGTCTTCCGATAGACTG	R (589-610)
	OJWB47	GTCGTGGGTAGTCAATCACTCAG	R and U5 (697-719)
	OJWB38	6-FAM-ATCCGAATCGTGGTCTCGCTGTTC-TAMRA	R (657-680)
Minus-strand synthesis	OJWB32	CCACAAGTTCAGCGTGTCC	GFP (3148-3166)
	OJWB33	CGTCGTCCTTGAAGAAGATGG	GFP (3366-3386)
	OJWB34	6-FAM-ATGTGGTCGGGGTAGCGGCTGAAGCAC-TAMRA	GFP (3283-3309)
Plus-strand strong stop	OJWB39	CAGTTCGCTTCTCGCTTCTGTTC	U3 (513-535)
	OJWB40	TGGGTAGTCAATCACTCAG	R and U5 (697-715)
	OJWB38	6-FAM-ATCCGAATCGTGGTCTCGCTGTTC-TAMRA	U5 (657-680)
Plus-strand extension	OJWB45	GCGCCAGTCTTCCGATAGACTG	R (589-610)
	OJWB48	ACAATCGGACAGACACAGATAAGTTG	Gag (807-832)
	OJWB38	6-FAM-ATCCGAATCGTGGTCTCGCTGTTC-TAMRA	R (657-680)
2LTR circles	OJWB45	GCGCCAGTCTTCCGATAGACTG	R (589-610)
	OJWB46	GGGCTCTTTTATTGAGCTCGGAG	U3 (549-571)
	OJWB38	6-FAM-ATCCGAATCGTGGTCTCGCTGTTC-TAMRA	R (657-680)

^aThe DNA intermediate of the MLV reverse transcription process that the primer/probe set detects.

^bDesignation given to each primer in a primer/probe set. Sets are listed in the following order: plus-strand primer, minus-strand primer, probe. The OJWB38 probe binds on the plus strand, and the OJWB34 probe binds to the minus strand.

^cSequence of the oligonucleotide written in the 5′-to-3′ orientation. Oligonucleotides used as Taqman probes are modified with a 6-FAM fluorescent probe at the 5′ end and a TAMRA quencher at the 3′ end.

^dThe functional elements of the viral genome the primer binds are denoted along with base pair position in the viral genome plasmid pLEGFP-C1. U3 is the unique 3′ region of the viral LTR, U5 is the unique 5′ region of the viral LTR, R is the repeat region of the LTR, and GFP correlates to the green fluorescent protein marker gene.