MOLgen

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Assay kit for the detection of DNA of Legionella pneumophila by PCR in real time.

“MOLgen DNA Legionella pneumophila S1 Kit” is intended for the detection of Legionella pneumophilaDNA in clinical specimens (oropharyngeal swabs, sputum, pleural fluid, bronchopulmonary lavage) and environmental samples (water samples, surface 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 the Internal Control Sample (IC) manufactured by Adaltis.

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

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 48 tests, including control samples.

NOTE: The extraction protocol and the PCR set-up procedure can be done in automated mode using the Adaltis EXTRAlab Instrument by executing the extraction and PCR set-up protocol preloaded in the instrument user interface.

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 pathogen’s DNA. Fluorescence signal increases due to the separation of fluorescence dye and quencher owing to 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 pathogen’s 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.