AffiPCR® CMV Quantitative Real Time PCR

Target Disease Type: Herpes Virus Infections

Specific Application: Cytomegalovirus (CMV)

Storage and Shipping : 4 weeks

CMV RT-PCR Quant (CE) V7-100/2FRT DataSheet

INTENDED USE

CMV Real-TM Quant PCR kit is an in vitro nucleic acid amplification test for qualitative detection and quantification of human cytomegalovirus (CMV) DNA in the clinical materials (peripheral blood plasma, amniotic fluid, cerebrospinal fluid (liquor), saliva, oropharyngeal swabs, urine samples, bronchoalveolar lavage, whole human blood, white blood cells, and viscera biopsy material) by using real-time hybridization-fluorescence detection.

PRINCIPLE OF PCR DETECTION

CMV determination by the polymerase chain reaction (PCR) with hybridization fluorescent detection includes three stages: DNA extraction from clinical samples, PCR-amplification of pathogen genome specific region, and real-time hybridization fluorescent detection. DNA is extracted from peripheral blood plasma, amniotic fluid, cerebrospinal fluid (liquor), saliva, oropharyngeal swabs, urine samples, bronchoalveolar lavage, whole human blood, white blood cells, and viscera biopsy material in presence of Internal Control (IC), which allows monitoring of analysis of each sample. Endogenous internal control (IC Glob - β-globin gene DNA) allows monitoring of PCR analysis stages (DNA extraction and PCR amplification), material sampling and storage adequacy. Then, CMV DNA is amplified using specific primers and polymerase (TaqF) .

β-globin gene DNA is a part of human genome DNA and it should be present in an adequate amount in DNA sample, obtained from the cells. There must be no less than 20 000 genomes per sample (DNA from 10 000 cells) . Internal Control (IC), added during the sample preparation from plasma, liquor, amniotic liquid, sputum, bronchial lavages and other cell free or low in DNA content materials, serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition, while endogenous IC (β-globine gene), present in all samples obtained from cells (whole blood, leucocytes, biopsy and autopsy material, saliva, swabs) allows not only to control analysis steps, but also to estimate sample handling and storage.

In real-time PCR, the amplified product is detected using fluorescent dyes. These dyes are linked to oligonucleotide probes which bind specifically to the amplified product during thermocycling. The real-time monitoring of the fluorescence intensities during the real-time PCR allows the detection of accumulating product without re-opening the reaction tubes after the PCR run. CMV Real-TM Quant PCR kit uses “hot-start”, which greatly reduces the frequency of nonspecifically primed reactions. “Hot-start” is guaranteed by separation of nucleotides and Taq-polymerase by using a chemically modified polymerase (TaqF) . Chemically modified polymerase (TaqF) is activated by heating at 95 ºC for 15 min.