Genotype analysis of anti b19 igm positive sera from brazil
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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

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
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



Alignment of 7 5 kda region of different parvovirus b19 isolates
Alignment of 7.5 kDa region of different parvovirus B19 isolates

MfeI site


Ns1 7 5 pcr
NS1/7.5 PCR

Nguyen et al, Virology 2002


Ns1 7 5 qpcr
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


Light cycler vs in house
Light cycler vs in house

  • Detect V9 and A6 sequences

  • Sensitivity of assay

  • < 1ge/uL

  • BUT

  • ?Confirmation of low positives

  • ? Sequence/genotype information


Pyrosequencing
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


Principle of method
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


B19 pyrosequencing
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



Validation of pyrosequence approach
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


B19 in brazil
B19 in Brazil

  • 50 IgM samples

  • 29 PCR positive

  • Range of concentrations: 102 -1010 ge/mL

  • All B19 genotype 1 (3 point mutations)



Acknowledgements
Acknowledgements

  • VRD:

  • Stuart Beard

  • Jayshi Ghandhi

  • Members of IDU

  • Angie Lackenby

  • Cath Arnold

  • Brazil:

  • Marilda Siqueira

  • SolangeOliveira


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