MOLgen

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“MOLgen RNA TBEV S1 Kit” is an assay kit for tick-borne encephalitis virus RNA detection by real-time PCR.

“MOLgen RNA TBEV S1 Kit”is intended for the detection of the tick-borne encephalitis virus (TBE) RNA using reverse transcription of viral RNA and real-time polymerase chain reaction (RT-PCR) method with fluorescence detection of amplified product.

The extraction of RNA from clinical specimens (whole blood, serum (plasma), leukocyte blood fraction, cerebrospinal fluid, biopsy materials and tick suspensions) can be performed using “Molgen Universal Extraction Kit”. When using NA extraction kits of other manufacturers it is highly recommended to use Internal Control sample (IC) manufactured by Adaltis.

The results of PCR analysis are taken into account in complex diagnostics of disease.

Set 1 is validated for use with block-type PCR cyclers: iQ5 iCycler, CFX96 (Bio-Rad, USA), DT-96 (DNA-Technology, Russia).

Set 1 contains reagents requiredfor 48 tests, including control samples.

Real-time PCR is based on the detection of the fluorescence produced by a reporter molecule, which increases as the reaction proceeds. Reporter molecule is a dual-labeled DNA-probe that specifically binds to the target region of pathogens DNA. Fluorescence signal increases due to the separation of fluorescence dye and quencher by Taq DNA-polymerase exonuclease activity during amplification. PCR consists of repeated cycles: temperature denaturation of DNA, primer annealing and complementary chain synthesis.

Threshold cycle value (Ct) is a cycle number at which the fluorescence generated within a reaction crosses the threshold and the fluorescence signal rises significantly above the background. Increased signal is due to the use of a DNA hybridization probe that is specific for the given DNA sequence: it binds to one of the DNA strands in the course of reaction and provides additional specificity of the method. A DNA probe consists of a fluorescence dye at the 5’-end and a fluorescence quencher at the 3’-end that significantly reduces the fluorescence intensity. During the polymerase synthesis of the complementary strand, the probe is cleaved from the 5’-end due to the 5’-3’ nuclease activity of Taq DNA polymerase, the quencher and the dye become separated, thus increasing the fluorescence signal due to accumulation of the reaction product. The detected fluorescence intensity depends on initial quantity of pathogens DNA template in the sample.

The use of Internal Control sample (IC) prevents generation of false negative results associated with possible loss of DNA template during specimen preparation. IC indicates if PCR inhibitors occur in the reaction mixture. IC should be added to each sample (including control samples) prior to the DNA extraction procedure. Amplification and detection of IC influence neither sensitivity nor specificity of the target DNA PCR.

Note: Internal Control sample is added to the specimen during NA extraction step and is used throughout the whole process of NA extraction, amplification, detection.