MOLgen - SARS-CoV-2 Real Time RT-PCR Kit

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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, salivary specimens 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 4 targets: 3 specific for SARS-CoV-2 (N gene, S Gene, 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), Cy5.5/Alexa Fluor (S gene) and CY5 (N gene).

The use of human beta actina 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 3 specific targets to SARS-CoV-2 (N , S 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-CoV-2 sequences (RdRP, E, S 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
• Refrigerator
• Microcentrifuge with rotor for 1,5 mL reaction tubes
• 1.5 mL microcentrifuge tube for extraction
• Proteinase K Qiagen a ≥ 20mg/mL - ≥ 600 U/mg code 19131 2 mL/vial code 19132 10 mL/vial
• Saliva collectors: Salivette® Sarstedt code 51.1534 or sterile polipropilene wide mouth centrifuge tubes with conical bottom
• Centrifuge
• Termoblok
• 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.

Ref: IFU

M. PERFORMANCE CHARACTERISTICS

ANALYTICAL SENSITIVITY

DIAGNOSTIC SENSITIVITY - ACCURACY - 100%

Diagnostics Sensitivity was in vitro assessed testing a Coronavirus SARS-CoV-NAT Positive Panel of 18 members tested using 2 Reference PCR methods.

ANALYTICAL SPECIFICITY (Cross-Reactivity)
Analytical Specificity was in vitro assessed testing related pathogens from the same genetic family and high priority organism likely in the circulating area.
Analytical Specificity was found 100%.

G. SPECIMENS COLLECTION AND STORAGE

• Collect fresh specimen of Nasopharyngeal swabs, Oropharyngeal swab, saliva and BALF from suspects. Refer to Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Patients Under Investigation (PUIs) for 2019 Novel Coronavirus (2019 nCoV).
• For collection methods refer to specimen collection devices manufacturer instructions.
• For the collection of the Nasopharyngeal and Oropharyngeal swabs, the manufacturer of the kit MOLgen SARS-CoV-2 Real Time RT-PCR, recommends the use of sterile collection system in liquid transport medium.
• For the collection of the saliva sample, the manufacturer of the MOLgen SARS-CoV-2 Real Time RT-PCR kit , recommends the use of undiluted saliva obtained by collection with Salivette® -Sarstedt code 51.1534 or sterile wide mouth centrifuge tubes in polypropylene and conical bottom (eg. Falcon ® 50 mL).
• Nasopharyngeal-Oropharyngeal swabs: Carefully take out the swab from package and quickly rotate it around two sides of fauces, throat and tonsila few times applying pressure to collect as much secretions as possible. Avoid touching tongue. Break the swab stick and put the head into sampling solution in specimen tubes. Screw the tube cap tightly to ensure no leakage.
• Saliva: Do not eat, drink and smoke 15 minutes before specimen collection. After saliva collection with Salivette® -Sarstedt code 51.1534, centrifuge for 2 minutes at 1000xg to obtain clear saliva samples in the conical tube. The saliva sample must have a volume at least 0.5 mL.
• BALF: Collect 3 ml of unprocessed BALF in sterile, dry and clean DNase/RNase free Cryotubes. Screw the tube cap tightly to ensure no leakage and seal the tube with film.

• Specimens can be stored at 2…8°C for up to 72 hours after collection or at -70°C for longer periods.

N. 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 the Kit more than 3 times.

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.