The GeneProof Cytomegalovirus (CMV) PCR Kit is an in vitro diagnostic medical device designed for professional use in laboratories, containing components for 25 or 100 reactions. It allows for the detection of CMV through real-time PCR, with specific storage and handling instructions to ensure stability and accuracy. The kit includes internal controls and is validated for various specimen types, with detailed protocols for sample collection, nucleic acid purification, and PCR setup.
The GeneProof Cytomegalovirus (CMV) PCR Kit is an in vitro diagnostic medical device designed for professional use in laboratories, containing components for 25 or 100 reactions. It allows for the detection of CMV through real-time PCR, with specific storage and handling instructions to ensure stability and accuracy. The kit includes internal controls and is validated for various specimen types, with detailed protocols for sample collection, nucleic acid purification, and PCR setup.
The GeneProof Cytomegalovirus (CMV) PCR Kit is an in vitro diagnostic medical device designed for professional use in laboratories, containing components for 25 or 100 reactions. It allows for the detection of CMV through real-time PCR, with specific storage and handling instructions to ensure stability and accuracy. The kit includes internal controls and is validated for various specimen types, with detailed protocols for sample collection, nucleic acid purification, and PCR setup.
The GeneProof Cytomegalovirus (CMV) PCR Kit is an in vitro diagnostic medical device designed for professional use in laboratories, containing components for 25 or 100 reactions. It allows for the detection of CMV through real-time PCR, with specific storage and handling instructions to ensure stability and accuracy. The kit includes internal controls and is validated for various specimen types, with detailed protocols for sample collection, nucleic acid purification, and PCR setup.
In vitro diagnostic medical device The kit has been manufactured according to the EC Directive 98/79/EC as an in vitro diagnostic medical device and it has been designed for professional use in specialized clinical and research laboratories.
The kit must be transported at a temperature of -20 °C or below. The kit will remain stable at least until the expiry date printed on the package, if the storage temperature is kept at (-20 ± 5)°C. The components are stable for a maximum of 5 repeated freezing / thawing cycles after the first use of a particular vial. Each component must be used before the expiry date or 30 days after the first use of a particular vial (whichever comes first).
TECHNICAL SPECIFICATIONCHNICAL SPECIFICATION
Target Sequence specific conservative DNA sequence of a single copy gene encoding the 4 IE antigen Analytical Specificity Human Cytomegalovirus (CMV), 100 % Analytical Sensitivity 122.594 IU/ml (on CMV NIBSC 09/162, manual extraction GeneProof PathogenFree DNA (LoD with the probability of 95 %) Isolation Kit), 165.237 IU/ml (on CMV NIBSC 09/162, automatic extraction croBEE 201A Nucleic Acid Extraction Kit) Diagnostic Specificity 90.67 % (CI95%: 81.15 % - 95.85 %) Diagnostic Sensitivity 92.86 % (CI95%: 64.17 % - 99.63 %) Linear Range 1010 - 102.5 IU/ml with precision of ± 0.5 log Dynamic Range 1010 - 122.594 IU/ml (using manual extraction GeneProof PathogenFree DNA Isolation Kit) 1010 - 165.237 IU/ml (using automatic extraction croBEE 201A Nucleic Acid Extraction Kit) Reporting Units IU/ml Validated Specimen plasma, serum, urine*, whole blood Quality Control regularly tested by QCMD and e.V. External Quality Assessment Panels Regulatory Status CE1023IVD
Quality management system is certified in compliance with the requirements of the standard ČSN EN ISO 13485 ed.2:2016 *NOTE: Validated only on manual extraction GeneProof PathogenFree DNA Isolation Kit.
INTERFERENCESFERENCES The interference testing was performed using positive and negative clinical specimens - plasma, serum and urine. The negative clinical specimens were spiked with CMV positive control at 3x LoD. The settings of pathological values for interference testing was performed according to CLSI guidelines EP7-A2 and hospital recommendations and guidelines (http://www.southend.nhs.uk/media/180421/pf_biochemistry_reference_intervals.pdf). Elevated levels of bilirubin (342 μmol/l), albumin (60 g/l), haemoglobin (2 g/l), urea (42.9 mmol/l), uric acid (1.4 mmol/l) and D-glucose (55 mmol/l) were tested in plasma and serum samples. The tested levels of endogenous interferents in urine (bilirubin, urea, uric acid, albumin, low pH, high pH level and D-glucose) were set according to the recommendations of Czech Society of Clinical Biochemistry.
The tested endogenous interferences were shown not to interfere with GeneProof Cytomegalovirus (CMV) PCR Kit with a significant level.
METHOD PRINCIPLEMETHOD PRINCIPLE
The PCR kit is designed for the detection of human Cytomegalovirus (CMV) by the real-time Polymerase Chain Reaction (PCR) method. The CMV detection consists in amplification of a specific conservative DNA sequence of a single-copy gene encoding the 4 IE antigen and in the measurement of fluorescence increase. The CMV presence is indicated by the FAM fluorophore fluorescence growth. An Internal Control (IC) is a part of the PCR kit, controlling both possible inhibition of the PCR and also the DNA extraction process quality. IC positive amplification is detected in the HEX fluorophore fluorescence channel. The detection kit takes advantage of the “hot start” technology, minimizing nonspecific reactions and assuring the maximum sensitivity. A Ready-to-Use Master Mix contains uracil-DNA-glycosylase (UDG), eliminating possible contamination of the PCR by amplification products. The kit is designed for in vitro diagnostics and provides qualitative and quantitative detection.
USER MANUAL SAMPLING AND SAMPLE STORAGE Sampling of all sample types, except for blood, should be performed into sterile tubes without any transportation media and the samples should be transported within 12 hours at the temperature (2 – 8) °C. It is necessary to sample up to 2 ml of body fluid samples (plasma, serum, urine, whole blood). Blood sampling: a sample of uncoagulable peripheral blood should be sampled into EDTA and transported into the laboratory at the temperature (2 – 8) °C within 24 hours. Urine samples are tested in case of glomerulonephritis symptoms. In case of longer storage keep all samples frozen at the temperature below -10 °C.
NUCLEIC ACID PURIFICATION
Nucleic acid extraction should be performed by extraction kits available on the market according to the protocols for the particular clinical material extraction. The manufacturer recommends the following products: GeneProof PathogenFree DNA Isolation Kit croBEE 201A Nucleic Acid Extraction Kit
Internal control (IC) should be added directly into a sample at the beginning of the extraction process so that in the end 1 μl of the resulting elution volume contains 0.1 μl of the IC: Elution volume 25 µl 50 µl 100 µl 200 µl
Internal Control 2.5 µl 5 µl 10 µl 20 µl
PCR SETUP 1. Gently vortex and briefly centrifuge the Master Mix and Calibrators' tubes.
2. Add 30 μl of Master Mix into PCR tubes.
3. Add 10 μl of the extracted nucleic acid sample or 10 μl of Calibrator into the individual PCR tubes and mix by pipetting. The total reaction mix volume will be 40 μl. It is necessary to keep all components at (2 – 8) °C during the PCR preparation. The isolate of negative extraction control with Internal Control should be used in each test. The negative clinical material, water or buffer can be used as a negative extraction control. The customer must use his own negative control. All 4 Calibrators must be used for setting the standard curve for quantitative detection.
4. Close the tubes, centrifuge shortly, insert them into the device and let them amplify according to the following PCR profile. Be very careful when handling the Calibrators or the clinical material; incorrect handling could result in contamination and the consequent impairment of the kit components! The manufacturer is not responsible for the kit impairment due to incorrect handling.
AMPLIFICATION PROFILE Step Temperature Time Data Collection Cycles
Hold 37 °C 2 min 1
Hold 95 °C 10 min 1
95 °C 5s
PCR 60 °C 40 s FAM + HEX 45
72 °C 20 s
INSTRUMENTSSTRUMENTS GeneProof Cytomegalovirus (CMV) PCR Kit is designed for use with real-time devices from various manufacturers:
croBEE Real-Time PCR System Mic qPCR Cycler
AMPLilab Real-Time PCR System QuantStudioTM 3 / 5 Real-Time PCR System Applied Biosystems 7300 / 7500 Real-Time PCR System Rotor-Gene 3000 / 6000 / Q AriaMx Real-Time PCR System SLAN® Real-Time PCR System BioQuant-96 Real-Time PCR System StepOne™ / StepOne PlusTM Real-Time PCR System CFX Connect™ / CFX96™/ Dx Real-Time PCR Detection System LightCycler® 2.0 / 480 LineGene 9600 / 9600 Plus
Required channels: FAM, HEX
GeneProof diagnostic kits are continually verified with various types of devices. Current list is available at www.geneproof.com or can be requested at support@geneproof.com
Use the following formula to calculate the virus concentration in IU/ml for manual extraction (using GeneProof PathogenFree DNA Isolation Kit):
SC - Sample concentration (IU/μl)
EV - Elution volume (μl) IV - Extraction volume (ml)
To easily calculate pathogen concentrations using manual or automated extraction, you can use the calculator at www.geneproof.com
WARNINGNING A single valid Instruction for Use for a specific kit is included in the package or to be requested for a particular lot from the manufacturer. Use only the combination of components from the given lot of the PCR kit. The kit should be disposed of after use according to the current legal regulations considering the fact that the kit does not contain any dangerous, infectious or toxic components that would be subject to special safety regulations, and the packaging materials are made of paper and polypropylene. If you have any questions, please contact our Customer care.
