MOLgen

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Reagent kit is intended for detection of Human papilloma virus (HPV) types 68, 73 and 82 DNA in clinical specimens by real-time PCR. The kit contains reagents for real-time PCR only.

“Molgen DNA HPV 68/73/82 S1 Kit” is intended for the differential detection of DNA of human papillomavirus (HPV) types 68, 73 and 82 in clinical specimens (epithelial cells swabs) using the method of real-time polymerase chain reaction (PCR) with fluorescence detection of amplified product.

The extraction of DNA from clinical specimens can be performed using the “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 S.r.l.

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

Set 1 can be used for quantitative determination of HPV types 68, 73 and 82 DNA when using the kit in set with “MOLgen DNA HPV HCR Genotype (quantitative) S1 Kit”.

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

Set 1 contains reagents requiredfor 96 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 dual-labeled DNA-probe, which specifically binds to the target region of pathogen DNA. Fluorescent signal increases due to the fluorescent dye and quencher separating by Taq DNA-polymerase exonuclease activity during amplification. PCR process consists of repeated cycles: temperature denaturation of DNA, primer annealing and complementary chain synthesis.

Threshold cycle value - Ct – is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal rises significantly above the background fluorescence. Increased fluorescence signal is due to the use of a specific for given CMV DNA sequence DNA hybridization probe that in the course of reaction binds with one of the DNA strands, also providing additional specificity of the method. DNA probe comprises of a fluorescent dye at the 5’ end and of fluorescence quencher at the 3’ end which significantly reduces the fluorescence intensity. During the polymerase synthesis of the complementary strand, due to the 5’-3’ nuclease activity of Taq DNA polymerase the probe is cleaved from the 5’-terminus and separation of the quencher and the dye occurs, resulting in the increase the fluorescence signal due to accumulation of the reaction product. Fluorescence intensity detected depends on initial quantity of pathogen DNA template in the sample.

The use of Internal Control (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 template should be added in each single sample (including control samples) prior to DNA extraction procedure. The amplification and detection of IC does not influence the sensitivity or specificity of the target DNA PCR.

Note: IC is a component of the NA extraction kit of MOLgen series. Internal Control is added to the sample during NA isolation step and is used throughout the whole process of NA extraction, amplification, detection.