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Certification of a Standard Reference Material for Cytomegalovirus (CMV)

Summary:

**Update**

As of the Summer of 2013, we discontinued distribution of SRM 2366 Cytomegalovirus (CMV) for DNA Measurements. In April of 2014, following in-house testing of the material, we released a letter notifying users and purchasers that the certified values for the number of amplifiable CMV genome copies listed in Table 1 of the Certified of Analysis (COA) are no longer valid. A re-analysis of the SRM components using digital PCR indicates evidence for variable degradation of the DNA construct. NIST recommends immediately discontinuing the use of SRM 2366. We truly regret that this SRM has degraded well before the expiration date listed on the COA. Accordingly, NIST will issue a refund and provide a free replacement once it is available. Instructions are provided in the letter


Cytomegalovirus (CMV) is commonly carried by the general population (50% to 80%). After the initial infection the virus generally remains latent in the individual. However, in immune compromised individuals, such as organ transplant patients or patients with HIV, severe and possibly life threatening disease can result. Congenital infections in infants can be the most severe. The treatment for cytomegalovirus is toxic and available only by intravenous administration. It is important to quantify the viral load of a patient in order to determine the time and length of treatment. Quantitative PCR is the current favored approach to measurement of viral load but there is no FDA approved method nor are there any reference materials available to the clinical community. About 50% of the clinical testing laboratories use commercial reagents and the other 50% used laboratory developed tests. Not surprisingly, there are great interlaboratory differences in the results of proficiency testing schemes.

 

Description:

The goal of this project is to provide a SI-traceable reference material for use by calibrant and reagent manufacturers in production of their own calibrants and standards. Clinical laboratories with in-house methods would be able to use the reference material for production of their own quality control materials which will be traceable to a NIST standard.  

It is non-trivial to produce a consistent Cytomegalovirus genome. Cytomegalovirus has a large genome and laboratory strains in culture in human fibroblasts have been shown to pack incomplete virus genomes in virus particles. An alternative is the use of a bacterial artificial chromosome (BAC) carrying the virus genome. NIST has acquired a BAC made from the Towne strain of Cytomegalovirus. The viral genome BAC was propagated in the bacteria, E. coli. The viral DNA will be certified for copy number and the sequence of selected regions will be released for informational value. Copy number will be determined using digital PCR. Regions to be sequenced include part or all of the following: UL34, UL54 (polymerase), UL55-56 (glycoprotein B), UL80, UL83 (phosphoprotein 65), UL97, UL122-126 (major immediate early and nearby sequence), UL132, and US17.


Major Accomplishments:

    • Acquired the Towne strain BAC as an alternative to viral culture
    • Developed protocols for producing viral DNA from bacterial cell culture
    • Sequenced the intended regions
    • Used digital PCR to determine the concentration in number of copies per microliter

Additional Technical Details:


PCR Detection-Specific Information:

CMV
http://www.newscentre.bham.ac.uk/images/CMV.jpg

Start Date:

June 1, 2007

End Date:

Ongoing

Lead Organizational Unit:

mml

Customers/Contributors/Collaborators:

Clinical testing community, Invitro device industry, Organizations: Quality Control for Molecular Diagnostics, Association for Molecular Pathology, National Institute of Biological Standards and Control (UK).

Contact

Dr. Peter M. Vallone
peter.vallone@nist.gov
301-975-4872