1. Intended Use
The Monkeypox Virus real time PCR kit is used for the detection of Monkeypox Virus in serum or
lesion exudate samples by using real time PCR systems.
2. Principle of Real-Time PCR
The principle of the real-time detection is based on the fluorogenic 5’nuclease assay. During the PCR reaction, the DNA polymerase cleaves the probe at the 5’ end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA. This cleavage results in the fluorescent signal generated by the cleaved reporter dye, which is monitored real-time by the PCR detection system. The PCR cycle at which an increase in the fluorescence signal is detected
initially is proportional to the amount of the specific PCR product. Monitoring the fluorescence intensities in real-time allows the detection of the accumulating product without having to re-open the reaction tube after the amplification.
3. Product Description
Monkeypox virus is the virus that causes the disease monkeypox in both humans and animals. Monkeypox virus is an Orthopoxvirus, a genus of the family Poxviridae that contains other viral species that target mammals. The virus is mainly found in tropical rainforest regions of central and West Africa. The primary route of infection is thought to be contact with the infected animals or their bodily fluids.The genome is not segmented and contains a single molecule of linear double-stranded DNA, 185000 nucleotides long.
The Monkeypox Virus real time PCR Kit contains a specific ready-to-use system for the detection of the Monkeypox Virus through polymerase chain reaction (PCR) in the real-time PCR system. The master contains reagents and enzymes for the specific amplification of the Monkeypox Virus DNA. Fluorescence is emitted and measured by the real time systems´ optical unit during the PCR.
The detection of amplified Monkeypox Virus DNA fragment is performed in fluorimeter channel 530nm with the fluorescent quencher BHQ1. DNA extraction buffer is available in the kit and serum or lesion exudate samples are used for the extraction of the DNA. In addition, the kit contains a system to identify possible PCR inhibition by measuring the 560nm fluorescence of the internal control (IC). An external positive control defined as 1×107 copies/ml is supplied which allow the determination of the gene load.
4. Kit Content
• All reagents should be stored at -20°C. Storage at +4°C is not recommended.
• All reagents can be used until the expiration date indicated on the kit label.
• Repeated thawing and freezing (> 3x) should be avoided, as this may reduce the sensitivity of
• Cool all reagents during the working steps.
• Reaction Mix should be stored in the dark.
6. Additionally Required Materials and Devices
• Biological cabinet
• Real time PCR system
• Desktop microcentrifuge for “eppendorf” type tubes (RCF max. 16,000 x g)
• Vortex mixer
• Real time PCR reaction tubes/plates
• Pipets (0.5 µl – 1000 µl)
• Sterile filter tips for micro pipets
• Sterile microtubes
• Disposable gloves, powderless
• Biohazard waste container
• Refrigerator and freezer
• Tube racks