NIST Standard Reference NIST Standard Reference Material Project: Pure DNA Material Project: Pure DNA

Standard for CytomegalovirusStandard for Cytomegalovirus

SoGAT XX SoGAT XX Warsaw, Poland June 13,2007Warsaw, Poland June 13,2007

Marcia HoldenMarcia Holden

NIST – The National Measurement NIST – The National Measurement Institution of the USAInstitution of the USA

the the NIST Laboratories, conducting research that advances the nation's conducting research that advances the nation's technology infrastructure and is needed by U.S. industry to continually improve technology infrastructure and is needed by U.S. industry to continually improve products and services;products and services;

the the Baldrige National Quality Program, which promotes performance excellence , which promotes performance excellence among U.S. manufacturers, service companies, educational institutions, and among U.S. manufacturers, service companies, educational institutions, and health care providers; conducts outreach programs and manages the annual health care providers; conducts outreach programs and manages the annual Malcolm Baldrige National Quality Award which recognizes performance Malcolm Baldrige National Quality Award which recognizes performance excellence and quality achievement;excellence and quality achievement;

the the Hollings Manufacturing Extension Partnership, a nationwide network of local , a nationwide network of local centers offering technical and business assistance to smaller manufacturers; centers offering technical and business assistance to smaller manufacturers; andand

the the Advanced Technology Program, which accelerates the development of , which accelerates the development of innovative technologies for broad national benefit by co-funding R&D innovative technologies for broad national benefit by co-funding R&D partnerships with the private sector. partnerships with the private sector.

NIST's core competencies:NIST's core competencies:

Measurement science Measurement science

Rigorous traceability Rigorous traceability

Development and use of standards Development and use of standards

Chemical Science and Technology LaboratoryChemical Science and Technology LaboratoryBiochemical Science DivisionBiochemical Science Division

Laurie Locascio, ChiefLaurie Locascio, Chief

DNA Measurements DNA Measurements GroupGroup

Cell and Tissue Cell and Tissue Measurements Group Measurements Group

Biospectroscopy Biospectroscopy GroupGroup

DNA Standard Reference MaterialsDNA Standard Reference Materials

Human DNA quantitation SRM 2372Human DNA quantitation SRM 2372

DNA Profiling SRMs 2391bDNA Profiling SRMs 2391b

Human Y chromosome DNA profiling SRM 2395Human Y chromosome DNA profiling SRM 2395

Mitochondrial DNA Sequencing SRM 2392-1Mitochondrial DNA Sequencing SRM 2392-1

Heteroplasmic Mitochondrial DNA SRM 2394Heteroplasmic Mitochondrial DNA SRM 2394

Oxidative DNA Damage Mass Spec SRM 2396Oxidative DNA Damage Mass Spec SRM 2396

Fragile X Syndrome SRM 2399Fragile X Syndrome SRM 2399

Why the need for a quantitative Why the need for a quantitative Cytomegalovirus (CMV) standardCytomegalovirus (CMV) standard

The monitoring of viral load is important in The monitoring of viral load is important in decisions on treatment for CMV disease decisions on treatment for CMV disease susceptible patients susceptible patients Quantitative Real Time PCR is rapidly Quantitative Real Time PCR is rapidly becoming the method of choice for viral becoming the method of choice for viral load determinations load determinations Variability in testing from laboratory to Variability in testing from laboratory to laboratory is related to the lack of laboratory is related to the lack of standardizationstandardization

CMV communityCMV community

A questionnaire was sent out to clinicians A questionnaire was sent out to clinicians and researchers to ascertain needsand researchers to ascertain needs

A workshop held at the Association for A workshop held at the Association for Molecular Pathology to discuss CMV Molecular Pathology to discuss CMV standardsstandards

A consensus was reached that NIST A consensus was reached that NIST should work on producing a standard for should work on producing a standard for CMV CMV

CMV DNA Standard Reference CMV DNA Standard Reference Material (SRM)Material (SRM)

Goal:Goal:

NIST will certify a CMV DNA SRM with an NIST will certify a CMV DNA SRM with an accurate and precise mass fraction accurate and precise mass fraction traceable to the SI and calculated genome traceable to the SI and calculated genome copy number for standardization of copy number for standardization of Quantitative Real Time PCR assays for Quantitative Real Time PCR assays for viral load determinationviral load determination

This CMV SRM is This CMV SRM is notnot designed to supply the testing designed to supply the testing community with all their standards needscommunity with all their standards needs

Many in the testing community are interested in matrix Many in the testing community are interested in matrix standards. standards.

There are vendors producing matrix standardsThere are vendors producing matrix standards Some laboratories are using calibrants produced in-house Some laboratories are using calibrants produced in-house

The interest here is in harmonization of the vendor The interest here is in harmonization of the vendor supplied materials as well as providing a standard that supplied materials as well as providing a standard that can be used to validate in-house produced standards. can be used to validate in-house produced standards. We would like to encourage vendors to make their We would like to encourage vendors to make their calibrants and standards material NIST-traceable using calibrants and standards material NIST-traceable using this SRM. this SRM. In this way, the SRM would contribute to reducing In this way, the SRM would contribute to reducing variability in testing from one laboratory to the next. variability in testing from one laboratory to the next.

Form of the CMV SRMForm of the CMV SRM

Pure CMV DNAPure CMV DNA

Cosmid-based synthetic DNA containing Cosmid-based synthetic DNA containing single copy CMV genes that are frequent single copy CMV genes that are frequent targets for Q-PCR assays, such as targets for Q-PCR assays, such as DNA polymeraseDNA polymerase Immediate Early GeneImmediate Early Gene Glycoprotein BGlycoprotein B

Issues with viral DNA isolated from Issues with viral DNA isolated from cell culturecell culture

Genome size - the proportion of full length Genome size - the proportion of full length to truncated genomes is dependent onto truncated genomes is dependent on Culture conditionsCulture conditions CMV strain and number of passagesCMV strain and number of passages

The ability to obtain sufficient quantity of The ability to obtain sufficient quantity of viral DNA for the standard as well as the viral DNA for the standard as well as the certification procedurecertification procedure

Cosmid CMV ConstructCosmid CMV Construct

Cosmids can hold up to 30 kb of insert Cosmids can hold up to 30 kb of insert which would allow the inclusion of multiple which would allow the inclusion of multiple genesgenesProduction of sufficient material should not Production of sufficient material should not be a problem. be a problem. Uniformity of the materials allows for Uniformity of the materials allows for easier purification and validation of sizeeasier purification and validation of sizeSmaller size, as compared with whole viral Smaller size, as compared with whole viral genome, will reduce DNA fragmentation genome, will reduce DNA fragmentation issueissue

Sources of materialSources of material

CMV genomic DNA – would not propagate CMV genomic DNA – would not propagate in-house, but would contract out for a in-house, but would contract out for a supply of CMV DNA with specified supply of CMV DNA with specified characteristics characteristics

Cosmid with inserted CMV single copy Cosmid with inserted CMV single copy genes – would produce and propagate in-genes – would produce and propagate in-house. PCR cloning of the individual house. PCR cloning of the individual genesgenes

Traceability of the CMV SRM to the SITraceability of the CMV SRM to the SI

Measurement of phosphorus content of purified DNAMeasurement of phosphorus content of purified DNA

Methodology is Inductively Coupled Plasma – Methodology is Inductively Coupled Plasma – Optical Emission Spectroscopy calibrated with a Optical Emission Spectroscopy calibrated with a NIST phosphorus SRMNIST phosphorus SRM

The High Performance version of ICP-OES gives The High Performance version of ICP-OES gives values with very small expanded uncertainties values with very small expanded uncertainties (0.1%)(0.1%) Ref: Traceable Phosphorus Measurements by ICP-OES Ref: Traceable Phosphorus Measurements by ICP-OES

and HPLC for the Quantitation of DNA. Holden et and HPLC for the Quantitation of DNA. Holden et alal. Anal. . Anal. Chem 79 (4): 1536-1541 2007Chem 79 (4): 1536-1541 2007

High Performance Inductively-Coupled High Performance Inductively-Coupled Plasma Optical Emission SpectroscopyPlasma Optical Emission Spectroscopy

99.0

99.5

100.0

Zr

Y

Ratio

0 5 10 15 20

“modern” ICP-OESwith solid-state detection

time-correlated internal standard

experimentdesign

drift correction

gravimetric sample handling &spike addition

drift

noise

10

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12345678910

UnknownSamples

Calibration

Materials

Results ±

Uncertainty

Possible second methodology for Possible second methodology for certification of the quantity of DNAcertification of the quantity of DNA

GC and/or LC Isotope dilution Mass GC and/or LC Isotope dilution Mass Spectroscopy of acid/enzyme-digested Spectroscopy of acid/enzyme-digested DNADNA NIST researchers Miral Dizdar and Pavel NIST researchers Miral Dizdar and Pavel

Jaruga Jaruga

Characterization of Cosmid CMV DNA Characterization of Cosmid CMV DNA or CMV genomic DNAor CMV genomic DNA

DNA purity – elimination of non-DNA DNA purity – elimination of non-DNA sources of phosphorussources of phosphorus

Proportion of mass fraction that is Proportion of mass fraction that is microbial DNA (Cosmid) or human DNA microbial DNA (Cosmid) or human DNA (viral culture)(viral culture)

Sequencing of the CMV genesSequencing of the CMV genes

Cosmid / genome size (Kb)Cosmid / genome size (Kb)

CMV SRM CMV SRM

The DNA will likely be in buffer solution rather The DNA will likely be in buffer solution rather than lyophilized and the SRM will be stored at than lyophilized and the SRM will be stored at -20-20ooC or -80C or -80ooCC

A lifetime will be determined for the material.A lifetime will be determined for the material.

Stability and homogeneity studies will be Stability and homogeneity studies will be conducted conducted

Interlaboratory studies will be conducted to Interlaboratory studies will be conducted to establish the commutability of the materialsestablish the commutability of the materials

In conclusionIn conclusion

Decision on the type of material needs to Decision on the type of material needs to be made soonbe made soonA certification process for quantitation of A certification process for quantitation of DNA has been validatedDNA has been validatedNIST has experience in DNA standards NIST has experience in DNA standards but we are not virologists, so we have an but we are not virologists, so we have an advisory committee of CMV researchers advisory committee of CMV researchers and cliniciansand clinicians

- and we hope to learn more from you- and we hope to learn more from you