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