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Genotype analysis of anti-B19 IgM positive sera from Brazil

Genotype analysis of anti-B19 IgM positive sera from Brazil. Kevin E Brown Immunisation and Diagnosis Unit Virus Reference Department Centre for Infections. Prevalence of variant B19. * Only detected in those born before 1973. Parvovirus B19 in Brazil.

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Genotype analysis of anti-B19 IgM positive sera from Brazil

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  1. Genotype analysis of anti-B19 IgM positive sera from Brazil Kevin E Brown Immunisation and Diagnosis Unit Virus Reference Department Centre for Infections

  2. Prevalence of variant B19 * Only detected in those born before 1973

  3. Parvovirus B19 in Brazil • Part of study of rash like illness in Brazil • Samples tested for measles, rubella, dengue and B19 • Preliminary/feasibility study • 50 B19 IgM positive • 5 from 10 different regions

  4. B19V Transcription Map

  5. Alignment of 7.5 kDa region of different parvovirus B19 isolates MfeI site

  6. NS1/7.5 PCR Nguyen et al, Virology 2002

  7. NS1/7.5 qPCR • Modified PCR • Quantitect SYBR Green (Qiagen) • Light cycler machine • 4 step cycling conditions: • 95C for 15 min • 45 cycles of: • 94C for 15s • 55C for 20s • 72C for 20s • 78C for 5s (data acquisition) G1 G3 G2

  8. Light cycler vs in house • Detect V9 and A6 sequences • Sensitivity of assay • < 1ge/uL • BUT • ?Confirmation of low positives • ? Sequence/genotype information

  9. Pyrosequencing • Sequencing by synthesis • Detection of pyrophophate • Fast and reliable • Ideal for short-read sequencing or mutation/SNP analysis • Requirement ss DNA • Use biotinylated primer

  10. Principle of Method Step 1 1 of 4 NTP added to mix If incorporated PPi produced Step 2 Sulphurlyase converts PPi to ATP Luciferase converts ATP to light Apyrase degrades dNTPs and ADP

  11. B19 pyrosequencing • SYBR green qPCR • Biotinylated reverse primer • PCR products saved • ssDNA by binding to streptavidin beads • DNA incubated with forward primer, polymerase, sulphurylase, luciferase and apyrase • Sequential addition of dNTP • Record light emmision with PyroMark ID (Biotage) • Identify sequence with Identifire software

  12. Pyrosequencing results

  13. Validation of pyrosequence approach • 2 variants identified • Both correctly identified by pyrosequencing • No. tested 57 routine samples (since Dec 2006) • All genotype 1 • 1 additional sample identified – suspected as variants

  14. B19 in Brazil • 50 IgM samples • 29 PCR positive • Range of concentrations: 102 -1010 ge/mL • All B19 genotype 1 (3 point mutations)

  15. 69 bone marrow samples PCR analysis only • 12 B19 positive; 7/12 variants

  16. Acknowledgements • VRD: • Stuart Beard • Jayshi Ghandhi • Members of IDU • Angie Lackenby • Cath Arnold • Brazil: • Marilda Siqueira • SolangeOliveira

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