Realtime cmv assay

The Roche CMV assay runs on the CAP/CTM system (Roche Molecular Diagnostics, Pleasanton, CA, USA) which consists of the COBAS AmpliPrep for sample preparation and the COBAS Taqman for realtime PCR. The Roche CAP/CTM CMV test uses primers and probes targeting a conserved region of the CMV genome (UL54, encodes DNA polymerase). This assay uses an automatic, magnetic bead nucleic acid isolation using the COBAS AmpliPrep system. The quantification linear range is from 137 to 9,100,000 IU/mL. The Abbott RealTime CMV assay (Abbott Molecular Inc., Des Plaines, IL, USA), sample preparation was carried out on m2000sp using the magnetic bead m2000 System DNA extraction kit, and amplification and detection of the UL34 and UL80.5 genes of CMV were conducted on the m2000rt using RealTime CMV kits. The quantification linear range of plasma is from 31 to 156,000,000 IU/mL using the Abbott RealTime CMV assay. Both assays were performed following the instructions of the respective manufacturers.

The Abbott RealTime CMV assay is carried out on an Abbott m2000 system. It has two automated test procedures, for processing plasma and whole-blood specimens. Sample preparation and PCR assembly are performed on an Abbott m2000sp instrument. Real-time PCR amplification and detection and result reporting are performed on an Abbott m2000rt instrument. The test procedures for plasma and whole blood share the same reagents for sample preparation, PCR amplification, and detection but differ in their sample input and elution volumes. DNA is extracted from 0.5 ml of a plasma sample and eluted in a 70-μl eluate or from 0.3 ml of a whole-blood sample and eluted in a 110-μl eluate. Plasma samples can be processed by either extraction procedure, but WB samples can be extracted only by the WB extraction procedure.
For both procedures, the total PCR volume is 60 μl (35 μl eluate and 25 μl master mix). An internal control (IC) is mixed into the lysis reagent before the initiation of sample preparation and is added to each specimen, calibrator, and control as a control for extraction efficiency and to monitor PCR inhibition.

The 1st WHO International Standard for Human Cytomegalovirus for Nucleic Acid Amplification Techniques (NIBSC code 09/162) comprises a whole-virus preparation of HCMV strain Merlin in Tris-HCl buffer with human serum albumin. It has been lyophilized in 1-ml aliquots. The lyophilized WHO IS was stored at −20°C prior to use. The WHO IS was reconstituted according to the manufacturer's instructions in 1 ml of nuclease-free water, to a nominal concentration of 5 × 10⁶ international units (IU)/ml. The reconstituted WHO IS was diluted in commercially available normal human EDTA-plasma, normal human EDTA-WB, or buffer (Tris-EDTA [TE] or TE with salmon testis DNA [Sigma-Aldrich] as a carrier). The normal human EDTA-plasma was purchased as a pool. The normal human WB was a pool of 4 units. Both plasma and WB pools were purchased from ProMedDx and were prescreened for the absence of CMV DNA by using the Abbott RealTime CMV assay. Dilutions were tested on the same day they were prepared or stored at −70°C for 7 days and then tested.

Dilutions of the standards and patient samples were extracted and amplified using the Abbott RealTime CMV assay on an Abbott m2000 system.

CMV DNA was analyzed using the Abbott RealTime CMV assay (Abbott, Chicago, IL, USA) from colorectal mucosa tissue of all patients at the Microbiology Laboratory of Bursa Uludag University Health Application and Research Centre. Additionally, plasma samples from 43 patients were also tested using the same assay. Viral nucleic acid extraction and amplification were performed using a fully automated Abbott m2000 Molecular Analyzer (Abbott, Chicago, IL, USA). The limit of quantitation for CMV DNA was <20 copies/mL (c/mL). To determine the IU/mL value in plasma, the kit manufacturer recommends multiplying c/mL values by 1.55.