CMV PCR Qnt w/reflex UL97 and UL54 Seq

Blood (EDTA Plasma)

The UW Clinical Virology Laboratory in the Department of Laboratory Medicine and Pathology incorporates the modified FDA approved Roche cobas® CMV Quantitative assay for the rapid detection of Cytomegalovirus (CMV) using real-time PCR amplification.

The lower limit of detection, LoD (the minimum virus level that gives a positive result in 95% of replicates), and the lower limit of quantitation, LLoQ, are both 34.5 IU/mL.

The Roche cobas® CMV assay uses primers specific for UL54 gene.

Serum, Sterile Body Fluids, Clear non-cellular fluid (e.g. BAL, or Washing) or Swabs in Viral Transport Media.

The performance characteristics of the test were determined by the University of Washington Department of Laboratory Medicine and Pathology. The modified version has not been cleared or approved by the US Food and Drug Administration

This order mnemonic will automatically reflex positive results to CMV Drug Resist, Rapid UL97 and UL54 [CMVDRS] with additional charge.

CMV Drug Resistance: Ganciclovir, Cidofivir and Foscarnet (UL54 and UL97 genes).

Mutations leading to CMV drug resistance have been described in the UL97 gene (phosphotransferase encoding region) and UL54 gene (DNA polymerase encoding region) of the CMV genome.

Mutations associated with resistance to Ganciclovir have been described in the UL97 gene. Genotypic analysis is performed by Sanger sequencing two overlapping amplicons of about 350kB of the 3’ end of the UL97 gene.

Mutations associated with resistance to Ganciclovir, Cidofivir and Foscarnet have been described in the UL54 gene. Genotypic analysis is performed by Sanger sequencing a 1kB region of the UL54 gene.

The minimum virus level that gives a sequencable PCR amplicon is 240 IU/mL (2.4 log IU/mL).

Specimens with undetectable CMV viral load are rejected.

This test was developed and its performance characteristics determined by the University of Washington Department of Laboratory Medicine and Pathology. It has not been cleared or approved by the US Food and Drug Administration.

Blood: EDTA plasma, PPT, Red top, SST

Sterile Body Fluid: 1 mL in sterile container.

Clear non-cellular fluid (e.g. BAL or Washing): 1 mL in sterile container.

Swabs in Viral Transport Media

Unacceptable samples: Plasma collected in sodium or lithium heparin.

Note: Separate specimens MUST be submitted when both Viral Serology and PCR testing are ordered.

Freshly drawn whole blood specimens may be held at 2 to 25°C for up to 36 hours prior to plasma/serum preparation through centrifugation.

Sterile Body Fluids, Clear non-cellular fluid (e.g. BAL, or Washing) or Swabs in Viral Transport Media may be held at 2 to 8°C for up to 48 hours.

Blood: Centrifugation upon arrival, remove plasma/serum from cells.

Plasma/serum specimens may be stored and/or transported:

• 2 to 8°C for up to 6 days
• -20°C ± 2°C for up to 12 weeks

Sterile Body Fluid: If CSF has cellular material, centrifuge & aliquot clear CSF. Freeze at -10°C or colder and ship on dry ice. If freezer and/or dry ice is unavailable, ship immediately at 2-8°C (ice pack). Sample may be stored at 2-8°C (ice pack) for up to 48 hours of collection. Samples stored at 2-8°C greater than 48 hours old may be rejected. Do not freeze/thaw.

Clear non-cellular fluid: Freeze at -10°C or colder and ship on dry ice. If freezer and/or dry ice is unavailable, ship immediately at 2-8°C (ice pack). Sample may be stored at 2-8°C (ice pack) for up to 48 hours of collection. Samples stored at 2-8°C greater than 48 hours old may be rejected. Do not freeze/thaw.

Outside Laboratory: Freeze at -10°C or colder and ship on dry ice. If freezer and/or dry ice is unavailable, ship immediately at 2-8°C (ice pack). Sample may be stored at 2-8°C (ice pack) for up to 48 hours of collection. Shipment time must not exceed 48 hours from collection if stored at 2-8°C. Samples stored at 2-8°C greater than 48 hours old may be rejected. Do not freeze/thaw.