NIST Standard Reference Material Project: Pure DNA Standard for Cytomegalovirus

1. NIST Standard Reference Material Project: Pure DNA Standard for Cytomegalovirus SoGAT XX Warsaw, Poland June 13,2007 Marcia Holden

2. NIST – The National Measurement Institution of the USA • the NIST Laboratories, conducting research that advances the nation's technology infrastructure and is needed by U.S. industry to continually improve products and services; • the Baldrige National Quality Program, which promotes performance excellence among U.S. manufacturers, service companies, educational institutions, and health care providers; conducts outreach programs and manages the annual Malcolm Baldrige National Quality Award which recognizes performance excellence and quality achievement; • the Hollings Manufacturing Extension Partnership, a nationwide network of local centers offering technical and business assistance to smaller manufacturers; and • the Advanced Technology Program, which accelerates the development of innovative technologies for broad national benefit by co-funding R&D partnerships with the private sector.

3. NIST's core competencies: • Measurement science • Rigorous traceability • Development and use of standards

4. Chemical Science and Technology LaboratoryBiochemical Science DivisionLaurie Locascio, Chief • DNA Measurements Group • Cell and Tissue Measurements Group • Biospectroscopy Group

5. DNA Standard Reference Materials • Human DNA quantitation SRM 2372 • DNA Profiling SRMs 2391b • Human Y chromosome DNA profiling SRM 2395 • Mitochondrial DNA Sequencing SRM 2392-1 • Heteroplasmic Mitochondrial DNA SRM 2394 • Oxidative DNA Damage Mass Spec SRM 2396 • Fragile X Syndrome SRM 2399

6. Why the need for a quantitative Cytomegalovirus (CMV) standard • The monitoring of viral load is important in decisions on treatment for CMV disease susceptible patients • Quantitative Real Time PCR is rapidly becoming the method of choice for viral load determinations • Variability in testing from laboratory to laboratory is related to the lack of standardization

7. CMV community • A questionnaire was sent out to clinicians and researchers to ascertain needs • A workshop held at the Association for Molecular Pathology to discuss CMV standards • A consensus was reached that NIST should work on producing a standard for CMV

8. CMV DNA Standard Reference Material (SRM) Goal: NIST will certify a CMV DNA SRM with an accurate and precise mass fraction traceable to the SI and calculated genome copy number for standardization of Quantitative Real Time PCR assays for viral load determination

9. This CMV SRM is not designed to supply the testing community with all their standards needs • Many in the testing community are interested in matrix standards. • There are vendors producing matrix standards • Some laboratories are using calibrants produced in-house • The interest here is in harmonization of the vendor supplied materials as well as providing a standard that can be used to validate in-house produced standards. • We would like to encourage vendors to make their calibrants and standards material NIST-traceable using this SRM. • In this way, the SRM would contribute to reducing variability in testing from one laboratory to the next.

10. Form of the CMV SRM • Pure CMV DNA • Cosmid-based synthetic DNA containing single copy CMV genes that are frequent targets for Q-PCR assays, such as • DNA polymerase • Immediate Early Gene • Glycoprotein B

11. Issues with viral DNA isolated from cell culture • Genome size - the proportion of full length to truncated genomes is dependent on • Culture conditions • CMV strain and number of passages • The ability to obtain sufficient quantity of viral DNA for the standard as well as the certification procedure

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

13. Sources of material • CMV genomic DNA – would not propagate in-house, but would contract out for a supply of CMV DNA with specified characteristics • Cosmid with inserted CMV single copy genes – would produce and propagate in-house. PCR cloning of the individual genes

14. Traceability of the CMV SRM to the SI • Measurement of phosphorus content of purified DNA • Methodology is Inductively Coupled Plasma – Optical Emission Spectroscopy calibrated with a NIST phosphorus SRM • The High Performance version of ICP-OES gives values with very small expanded uncertainties (0.1%) • Ref: Traceable Phosphorus Measurements by ICP-OES and HPLC for the Quantitation of DNA. Holden et al. Anal. Chem 79 (4): 1536-1541 2007

15. Results ± Uncertainty 100.0 Calibration Materials UnknownSamples 99.5 Zr 99.0 Y Ratio 1 3 4 5 6 7 8 9 10 1 2 2 3 4 5 6 7 8 9 10 High Performance Inductively-Coupled Plasma Optical Emission Spectroscopy “modern” ICP-OES with solid-state detection drift correction noise drift experiment design time-correlated internal standard gravimetric sample handling & spike addition

16. Possible second methodology for certification of the quantity of DNA • GC and/or LC Isotope dilution Mass Spectroscopy of acid/enzyme-digested DNA • NIST researchers Miral Dizdar and Pavel Jaruga

17. Characterization of Cosmid CMV DNA or CMV genomic DNA • DNA purity – elimination of non-DNA sources of phosphorus • Proportion of mass fraction that is microbial DNA (Cosmid) or human DNA (viral culture) • Sequencing of the CMV genes • Cosmid / genome size (Kb)

18. CMV SRM • The DNA will likely be in buffer solution rather than lyophilized and the SRM will be stored at -20oC or -80oC • A lifetime will be determined for the material. • Stability and homogeneity studies will be conducted • Interlaboratory studies will be conducted to establish the commutability of the materials

19. In conclusion • Decision on the type of material needs to be made soon • A certification process for quantitation of DNA has been validated • NIST has experience in DNA standards but we are not virologists, so we have an advisory committee of CMV researchers and clinicians - and we hope to learn more from you