Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

Table 1 Oligonucleotides used for EBV QPCR, beta-globin detection, construction of plasmid and PCR thermal cycling conditions.

Target	Primer Name	Oligonucleotide Sequence 5'-3'	Amplicon Length	GenBank Accession (position)	Reference	Optimised PCR Thermal Cycling Conditions
EBNA-1	QP1	GCC GGT GTG TTC GTA TAT GG	213 bp	AJ507799 (97174-97386)	Stevens et al, 1999	95°C initial denaturation for 10 mins; 58°C annealing
	QP2	CAA AAC CTC AGC AAA TAT ATG AG
BHRF-1	EA-1F	GGA GAT ACT GTT AGC CCT G	208 bp	AJ507799 (42105-42312)	Custom	98°C initial denaturation for 13 mins; 60°C annealing
	EA-2R	GTG TGT TAT AAA TCT GTT CCA AG
Plasmid construct (randomised primers in bold)	EA-F	CTA TAT GTC TGC TTA CTC CGG CG /G GAG ATA CTG TTA GCC CTG	554 bp	N/A	Custom	95°C initial denaturation for 10 mins; 55°C annealing
	EB-R	CGC CGG AGT AAG CAG ACA TAT AG /CAA AAC CTC AGC AAA TAT ATG AG				95°C initial denaturation for 10 mins; 55°C annealing
Beta-Globin TAL57	BG-1F	TAG CAA CCT CAA ACA GAC ACC A	247 bp	EU760960 (171-417)	Custom	95°C initial denaturation for 10 mins; 61°C annealing
	BG-1R	CAG CCT AAG GGT GGG AAA AT

Abbreviations: EBNA-1, Epstein-Barr virus nuclear antigen-1; BHRF-1, BamHI fragment H rightward open reading frame-1; mins, minutes

ISSN: 1743-422X