Evaluation of Cloned DNA for B19 Evaluation of Cloned DNA for B19 GenotypesGenotypes

Dr Jacqueline Fryer, Division of Dr Jacqueline Fryer, Division of VirologyVirology

NIBSCNIBSC

Genetic Diversity of Parvovirus Genetic Diversity of Parvovirus B19: Analysis of the NS1/VP1 B19: Analysis of the NS1/VP1

RegionRegion

A6

Servant et al., J. Virol. 2002

Plasmid InformationPlasmid Information

PlasmidPlasmid GenotypeGenotype Insert sizeInsert size

(bp)(bp)VectorVector SourceSource

pB19-N8pB19-N8 11 52245224 pCR-Blunt pCR-Blunt II-TOPO II-TOPO ((Kan)Kan)

Zhi Zhi et alet al. . 20042004

pA6-cl8pA6-cl8 22 49154915 pCR-Blunt pCR-Blunt II-TOPO II-TOPO ((KanKan))

Nguyen Nguyen et et al.al. 2002 2002

pV9pV9 33 50285028 pcDNA2.1pcDNA2.1

((AmpAmp))Nguyen Nguyen et et alal. 1999. 1999

pD91.1pD91.1 33 50355035 pcDNA3.1pcDNA3.1

((AmpAmp))Servant Servant et et alal. 2002. 2002

Evaluation of PlasmidsEvaluation of Plasmids

Transformed each plasmid & maxi preps Transformed each plasmid & maxi preps grown up for each clonegrown up for each clone

DNA sequence analysis of insert/vector DNA sequence analysis of insert/vector region determinedregion determined

Restriction enzyme digestions of clonesRestriction enzyme digestions of clones

Analysis by consensus real-time PCR assay Analysis by consensus real-time PCR assay

Analysis of Plasmid DNAs by Analysis of Plasmid DNAs by Consensus Assay for Parvovirus Consensus Assay for Parvovirus

B19B19

Distribution and Use of Clones to Distribution and Use of Clones to OMCL Network and ManufacturersOMCL Network and Manufacturers

To be distributed through the EDQM PTS To be distributed through the EDQM PTS for parvovirus B19 DNAfor parvovirus B19 DNA

To be used for the validation of assays & To be used for the validation of assays & material not to be distributed beyond the material not to be distributed beyond the recipients laboratoryrecipients laboratory MTA with NIH for pB19-N8, pA6MTA with NIH for pB19-N8, pA6 Hôpital Armand Trousseau permission to Hôpital Armand Trousseau permission to

supply pV9 & pD91.1supply pV9 & pD91.1