Hepatitis B

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MOLgen RNA HAV1 S1 Kit is intended for detection of hepatitis A virus (HAV) RNA by real-time RT-PCR. The kit contains reagents for real-time RT-PCR only.

“Molgen RNA HAV S1 Kit” is intended for the detection of hepatitis A virus (HAV) RNA in clinical specimens (blood serum and plasma, faeces) and water specimens using the method of reverse transcription of viral RNA with subsequent cDNA amplification by real-time polymerase chain reaction (RT-PCR) with fluorescence detection of amplified product.

The extraction of RNA from clinical specimens can be performed using “MOLgen Universal Extraction Kit” (manufactured by Adaltis S.r.l.). When using NA extraction kits of other manufacturers it is highly recommended to use Internal Control sample (IC) manufactured by Adaltis.

The results of RT-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 cDNA. 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 cDNA, 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 cDNA sequence: it binds to the cDNA 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 cDNA template in the sample.

The use of Internal Control sample (IC) prevents generation of false negative results associated with possible loss of RNA/cDNA 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 RNA extraction procedure. Amplification and detection of IC influence neither sensitivity nor specificity of the target cDNA 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.