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Primer & Probe Design Affects Ability to Detect and Quantify Variant Viruses

This study investigates how the design of primers and probes affects the detection and quantification of variant viruses, specifically Parvovirus B19 genotypes. We validate the VP1 TaqMan assay and utilize SYBR Green detection, analyzing samples with a concentration of 106 copies per reaction. The research emphasizes the need for high fidelity in probe design to improve detection efficiency among different genotypes. Variability in quantification metrics reinforces the necessity for standardizing materials in quantitative assays to ensure accurate viral load measurements.

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Primer & Probe Design Affects Ability to Detect and Quantify Variant Viruses

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  1. 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 A6 a t t c g ca a V9 g c C t a ag g D91.1 g c C t a ag a B19 a t C t g ca g Consensus ATCATTTTCA -AG-CATGGA CAGTTAT-TG ACCACCCCCA TGCCTTATCA -CCAGTA-CA GT--T-CAGA GA CAGTTATCTG ACCACCCCCACCAGTAGCA GTCATGCAGA A6 g g g a a c V9 a g A a a t D91.1 a a A a g c B19 a g A g a c Consensus ACCTAGAGGA GAA-ATGCAG TATTATCTA- TGAAGACTTA CACAAGCCTG GGC-AGTTAG C-T-CAA-TA ACCTAGAGGA GAACAAGCCTG GGCAAGTTAG C • Aim to validate VP1 TaqMan/ ABI 7700Aberham J. Virol. Meth. 92, 183-91 (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 SYBR Green Detection 106 copies/reaction A6 G CA a V9 aag G D91.1 aag a B19 G CA G Consensus CCAGTA-CA GT--T-CAGA ACCTAGAGGA GA Probe CCAGTAGCA GTCATGCAGA ACCTAGAGGA GA Aberham J. Virol. Meth. 92, 183-91 (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 D91.1 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

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