MOLgen

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“Molgen DNA HPV HCR genotype S1 Kit” is an assay kit for the differential determination of DNA of Human Papillomavirus types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 of high cancer risk by Real-Time PCR method.

“Molgen DNA HPV HCR genotype S1 Kit” is designed for differential determination of DNA of human papillomavirus (HPV) types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59 of high cancer risk isolated from clinical specimens using extraction kit: “MOLgen Universal Extraction Kit”. Assay is based on real-time polymerase chain reaction (PCR) method with fluorescent detection of amplified product.

The kit contains reagents requiredfor 96 tests, including control samples.

The kit is validated for use with iQ iCycler, iQ5 iCycler (Bio-Rad, USA). The kit is compatible with other real-time PCR systems such as CFX96 (Bio-Rad, USA) and DT-96 (DNA-Technology, Russia).

“Molgen DNA HPV HCR genotype S1 Kit” is designed for the analysis of clinical materials (scrapings of the epithelial cells, semen, prostatic fluid, urine).

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 HPV 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 (RMM1, 2, 3). 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.

To analyze each sample for the content of HPV DNA half of the strip is used - the amplification is carried out in four tubes (RMM1, RMM2, RMM3, RMM4) for detection of DNA of three of the 12 HPV types in each tube (see Table 1).

Table 1

In order to validate the quality of sampling and improve the reliability of results of HPV HCR DNA detection, the content of human DNA (β-actin) is additionally estimated in the analyzed samples (RMM4).