SARS-CoV2 - 3 Gene

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MOLgen SARS-CoV-2 Real Time RT-PCR Kit is used for the qualitative detection of Novel Coronavirus (SARS-CoV-2), by Reverse Transcription (RT) and Real Time Polymerase Chain Reaction (PCR)  from RNA extracted from human respiratory  specimens such as nasopharyngeal swabs, oropharyngeal swabs and bronchoalveolar lavage fluid (BALF).

Coronaviruses are a large family of respiratory viruses, some causing illness in human and others circulating among animals such as camels, cats and bats from common cold to severe diseases such as Middle East Respiratory Syndrome (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS-CoV).

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a new strain of coronavirus emerged from Wuhan, China in December 2019.
The infection with SARS CoV2 virus causes Coronavirus disease (COVID-19) where "CO" stands for crown, "VI" for viruses, "D" for disease and “19'" indicates the year in which it occurred.

COVID-19 symptoms are fever, cough , pneumonia and Severe Acute Respiratory Syndrome (SARS).

MOLgen SARS-CoV-2 Real Time RT-PCR Kit is intended for the detection of the New Coronavirus SARS-CoV-2 RNA using reverse transcription of viral RNA and real-time polymerase chain reaction (RT-PCR) method with fluorescence detection of amplified product.

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 the 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. The 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. The 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 the initial quantity of pathogen’s cDNA template in the sample.

The primer and probe set is designed to detect SARS-CoV-2 identifing 3 targets: 2 specific for SARS-CoV-2 (N gene and E gene) and 1 common for sabercoviridae (RdRp gene).

The detection of amplified virus RNA fragment is performed in fluorimeter channel FAM (RdRp gene), ROX/Texas Red (E gene) and CY5 (N gene).

The use of human beta actin as Internal Control (IC) on HEX channel ensure the monitoring correct amplification and cellularity of the samples.
The results of PCR analysis are taken into account in complex diagnostics of disease. The kit is validated for use with: AMPLIlab (Adaltis), CFX96 (Bio-Rad).

Applications Kit for Novel Coronavirus (SARS-CoV-2) detection using real-time PCR based on the use of Dual Probes

The kit contains reagents required for 100 reactions.

1. MOLgen SARS-CoV-2 Master Mix Ready to use reagent.
It contains reagents for reverse transcription and amplification.

2. MOLgen SARS-CoV-2 Probe Mix Ready to use reagent.
It contains primers and probes which are premixed to the working concentrations to identifying 2 specific targets to SARS-CoV-2 (N and E genes) 1 common target for sabercoviridae (RdRp gene) and 1 for human beta actin.

3. MOLgen SARS-CoV-2 Positive Control Ready to use control.
It contains plasmids with SARS-CoV2 sequences (RdRP, E and N genes) plus an Internal Control (IC).
The Internal Control is a plasmide with sequence of human beta actin.

4. MOLgen SARS-CoV-2 Negative Control Ready to use control.
Negative Control should be included at every run as it indicates that reagents have not been contaminated.

E. MATERIALS AND DEVICES REQUIRED BUT NOT SUPPLIED

• Nucleic Acid extraction kit intended for isolation of RNA (MOLgen Universal Extraction Kit (Adaltis) Ref. ME188830).
• Real-time PCR cycler
• Biosafety Cabinet
• RefrigeratorAdaltis Srl 4/8 936MESARS-CoV-2/Rev. Date: 210215_CE_(EN)
• Microcentrifuge with rotor for 1,5 mL reaction tubes
• 1.5 mL microcentrifuge tube for extraction
• Plate shaker
• Calibrated adjustable pipettes
• Disposable pipette tips with aerosol filter
• Strip Tubes and Caps (0.2 mL) or PCR Tubes (0.2 mL) or 96 well plate with optical caps or films.
• Disposable medical non-sterile powder-free gloves
• Magnetic rack for tubes
• Biohazard waste container

NOTE: The extraction protocol with MOLgen Universal Extraction Kit (Adaltis) Ref. ME188830 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.

STORAGE AND TRANSPORTATION

• Transport the kit at -15°C…-25°С.
• Store the kit in the manufacturer’s packaging at -15°C…-25°С for the entire shelf life.
• Do not freeze/Thaw the Kit more than 3 times.

WARNING AND PRECAUTIONS

This kit has to be used by skilled and properly trained technical personnel only, under the supervision of a medical doctor responsible of the laboratory.

• To obtain reliable results, strictly follow this Instruction      for use provided with the kit.

• Read carefully the Safety Data Sheet (SDS) before product use.

• Do not use the components from the Kits of different lots.

• For components avoid more than 3 thawing- freezing cycles.

• All the personnel involved in performing the assay  have to  wear protective laboratory clothes, talc-free gloves and glasses.All personnel involved should be trained in biosafety procedures, as recommended by the Center for Disease Control, Atlanta, U.S. and reported in the National Institute of Health’s publication: “Biosafety in Microbiological and Biomedical Laboratories”.

• When handling the kit, follow the national safety requirements for working with pathogens.

• Treat all specimens as potentially infective using  safe laboratory procedure. Refer to Interim Laboratory Biosafety Guidance for Handling and Processing Specimens Associated with 2019-nCoV.

• To prevent cross-contamination it is recommended to have  dedicated areas for sample preparation (RNA extraction) and PCR test (setting up PCR reaction and thermocyclers).

• Wear protective disposable gloves, laboratory coats and eye protection when handling specimens and kit reagents.

• Every workplace must be provided with its own set of variable-volume pipettes, necessary auxiliary materials and equipment. It is prohibited to relocate them to other workplaces.

• To conduct real-time amplification reaction with PCR products detection, use only disposable tips with aerosol filters.

• Do not pool reagents from different lots or from different vials of the same lot.

• Dispose unused reagents and waste in accordance with country, federal, state and local regulations.

• Do not use the kit after the expiration date. • Do not smoke,drink eat or apply cosmetics in areas in which specimens or Kit components are handled.