Development of a NIST Standard Reference Material for Cytomegalovirus

Transcription

1 Development of a NIST Standard Reference Material for Cytomegalovirus Marcia Holden, Ross Haynes, Margaret Kline, John Butler (with help from David Duewer (NIST) and Steve Ellison (LGC)) Group, Biochemical Science Division National Institute of Standards and Technology, Gaithersburg, MD USA SoGAT Clinical Diagnostics II 2009

2 CMV Standard Reference Material Reference materials for quantitative realtime PCR assays for viral load testing Type of material - pure viral DNA in buffer CMV DNA, Towne strain, cloned into a bacterial artificial chromosome (Towne 147) The BAC/CMV DNA is circular and is essentially a very large plasmid and is purified by a process that is similar to plasmids

3 CMV genome cloned into a bacterial artificial chromosome, what are the advantages - - Consistent genome size Due to the large genome size of CMV (240,000 base pairs), it is difficult to obtain consistent genomes from culture of intact virus in the laboratory BACs are propagated in bacterial culture

4 147 Towne BAC BAC DNA Can accommodate up to 300,000 base pairs Contains the F factor which maintains 1-2 copies per cell and the elements that guide partitioning of copies between dividing cells BACs are considered to be stable. DNA has been removed from the CMV viral genome to accommodate the BAC related DNA BAC DNA replaces US1 to US15 in the CMV genome (about 10,000 bp) Induction segment (inducible origin of replication) replaces UL 147 DNA that has been removed is not in the regions used for amplification

5 Certification of the CMV DNA What properties of the CMV DNA will be certified - - DNA sequence of regions of the CMV genome that are used as targets of PCR amplification for viral load testing Copy number by direct measurement

6 DNA sequencing To date the sequencing of specific regions has indicated that the Towne BAC and the sequence in GenBank are the same. The following regions have been sequenced UL54 (DNA pol), UL55 (glycoprotein B), UL83 (PP65), UL122 (IE), UL123 (MIE), US17 If there are other regions that are relevant to PCR for viral load testing, please let us know and we will add those regions.

7 Certification of the CMV DNA - genome copy counting Digital PCR Quantify the amount of DNA (copies/volume) by counting amplification from single molecules Nano scale reactions (as little as 6 nl) Thousands of replications/assay repeated with multiple assays targeting regions on the CMV genome DNA concentration where there is 0-1 copy per reaction chamber Traceable to the SI via the Mole/Unity New tool, with active research at NMIs like NIST on validation of this approach

8 Digital PCR Pressurized valve Samples: 1-12 Water: H H X X H 765 individual chambers / panel 12 panels/chip Pressurized valve

9 Preparation of the SRM Three levels of concentration of DNA Packaging in Teflon tubes Certification for copy number/volume will be done on each of the concentrations Monitoring for homogeneity and stability for the life of the SRM (5 years from time of issue) We currently have a stock that we have been monitoring for 6 months at three temperatures

10 QCMD CMV Proficiency testing program for 2009 Paul Wallace Calum Scott William Mackay NIST was invited to submit a sample of the SRM candidate material for analysis by the participants in the 2009 CMV program. DNA in buffer was prepared from our stock and packaged in tubes Assigned a value to the material of 3.8 M copies / ml. using digital PCR (Expanded uncertainty of 3.4 M to 4.2 M copies/ml). Log10 = 6.58 Tubes were sent to QCMD for packaging with the QCMD test samples. Monitored stability and homogeneity for the time frame of the program and beyond. Participants were asked to add the DNA solution directly to their assay and run the assay in triplicate. The handling of the sample was different from the standard samples 2009 QCMD CMV PT program

11 Multiplicative Standard Deviation, Copies per ml Relative Area, #Bin / (#Total Δx) Commercial In-House Conventional Total Gaussian Model Median: = Copies per ml MADe: = 3.8 u (Median): = Median s repeat 1.09: Commercial 1.10: In-House 1.06: Conventional S interlab : QCMD CMV PT program log 10 (Copies per ml)

12 Relative Area, #Bin / (#Total Δx) Kernal Density Fit Centered Histogram Kernal Density Model A Model B FWHM Log 10 (Copies per ml) 2009 QCMD CMV PT program

13 Relative Area, #Bin / (#Total Δx) Kernal Density Fit Centered Histogram Kernal Density Model A Model B FWHM QCMD CMV PT program Log 10 (Copies per ml)

14 Density gaussian mixture N = 153 Bandwidth = Group 1: Center = 5.85, s = 1.31 Group 2: Center = 6.49, s = QCMD CMV PT program Maximum likelihood routine

15 Density gaussian mixture N = 153 Bandwidth = Group 1: Center = 5.645, s = 1.44 Group 2: Center = 6.16, s = 0.21 Group 3: Center = 6.79, s = QCMD CMV PT program Maximum likelihood routine

16 Technology Group Mean Log10 Std Dev Range for Copies/mL Copies/mL log 10 1 Std Dev # data sets RT Commercial 2.96 M M M 74 RT In-house 1.44 M M M 74 Conventional Commercial 1.49 M M M 5 Maximum Likelihood routine 2 Gaussian Mixture 0.71 M M M M M M 84 3 Gaussian Mixture 0.44M M M M M M M M M 52 U Digital PCR 3.8 M M M

17 Distribution of datasets into 4 groupings/bins Bin s Log10 Copies/mL Copies/mL Comm-RT In house RT Total Lo <5.738 < 0.5M Gp M M Gp M M Hi >7.124 > 13.3M

18 Towne AD169 AD169 Toledo Merlin (clinical) (clinical) 2009 Molecular Virology Workshop

19 Towne AD169 AD169 Toledo Merlin (clinical) (clinical) 2009 Molecular Virology Workshop

20 Distribution of datasets within the four groupings breakdown of commercial reagents CutPoints % Code # < > Low Gp1 Gp2 High %Out In-house RTIH QIAGEN Nanogen Argene Roche-LightCycler Roche-COBAS Cepheid astra ATQ Diagenode GeneProof LightUp Sum All 19 RTC 2009 QCMD CMV PT program

21 Groupings based on use/or not of external calibrants External Calibrants used Bins No Yes Lo Group Group Hi 6 9 Total QCMD CMV PT program

22 Breakdown of groupings by commercial reagents External Calibrant used Assay Used Group Yes No Argene Lo 1 0 Hi Nanogen Lo 1 1 Hi Qiagen Lo 0 1 Hi Lab developed Lo 11 9 Hi QCMD CMV PT program

23 Summary Progress has been made in the production and validation of a candidate reference material with plans to finish the process in 2010 Participation in the 2009 QCMD CMV PT program has demonstrated that the material can be measured by a variety of CMV viral load assays, both commercial and laboratory-developed tests (but not all)