1. Sally Baylis, Piotr Grabarczyk & Jacqueline Fryer
Primer & Probe Design Affects Ability to Detect and Quantify Variant Viruses
Sally Baylis, Piotr Grabarczyk &
Jacqueline Fryer

2. Detection of Parvovirus B19 Genotypes with SYBR Green
A a t t c g ca a
V g c C t a ag g
D g c C t a ag a
B a t C t g ca g
Consensus ATCATTTTCA -AG-CATGGA CAGTTAT-TG ACCACCCCCA TGCCTTATCA -CCAGTA-CA GT--T-CAGA
GA CAGTTATCTG ACCACCCCCA CCAGTAGCA GTCATGCAGA
A g g g a a c
V a g A a a t
D a a A a g c
B a g A g a c
Consensus ACCTAGAGGA GAA-ATGCAG TATTATCTA- TGAAGACTTA CACAAGCCTG GGC-AGTTAG C-T-CAA-TA
ACCTAGAGGA GA ACAAGCCTG GGCAAGTTAG C
Aim to validate VP1 TaqMan/ ABI 7700 Aberham J. Virol. Meth. 92, (2001) for detection of genotypes 3
Clone fragments of DNA from B19 (IS 99/800), A6, V9 and D91.1
Sequence to confirm identity of templates
Use in assays at 106 copies of each genotype /PCR
Assay performed on Roche LightCycler v1.0

3. Detection of Parvovirus B19 Genotypes with TaqMan Probe with Incomplete Homology
A G CA a
V a ag G
D a ag a
B G CA G
Consensus CCAGTA-CA GT--T-CAGA ACCTAGAGGA GA
Probe CCAGTAGCA GTCATGCAGA ACCTAGAGGA GA
SYBR Green Detection
106 copies/reaction
Aberham J. Virol. Meth. 92, (2001) TaqMan VP1
Annealing at 60ºC Annealing at 56ºC
B19
B19
TaqMan Detection
106 copies/reaction
TaqMan Detection
106 copies/reaction A6 A6 V9 V9
D91.1
D91.1
NTC
NTC
No Ct values detectable for D Ct values detectable for D91.1
Efficiency slightly variable between genotypes

4. Need for materials to standardise quantitative assays – what are the right materials?
- Genotype 2 virus, 98.3% identity with A6, 6.2 logs
- Genotype 3 virus, 5 logs