GEN-MPV-PCR-150T

Monkeypox Virus Real Time PCR Kit 150 Tests/Kit CE Version

We as rapid diagnostics have the PCR tests for Monkey pox develloped by Genprice's our molecular division.

• For tests in the US please contact the CDC
• Patients with symptoms in Europe can be tested in the Institute of tropical medicine in Antwerp, Belgium

Monkeypox Virus Real Time PCR Kit

User Manual

• For Research Use Only
• Cat. No.: PDPS-AR064
• Lot. No.: (See product label)

1. Introduction

Genprice Monkeypox Virus real time PCR kit is used for the
detection of Monkeypox Virus in serum or lesion exudate samples.

The kit contains a specific ready-to-use system for Monkeypox Virus
detection through Reverse Transcription Polymerase Chain
Reaction (RT-PCR) in the Liferiver real-time PCR system.

2. Intended Use

The Monkeypox Virus real time PCR kit is a test for the detection of
Monkeypox Virus in serum or lesion exudate samples in real time
PCR systems.

3. 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 (Ct) is proportional to the
amount of the specific PCR product. Monitoring the fluorescence
intensities during Real Time allows the detection of the accumulating
product without having to re-open the reaction tube after the
amplification.

4. Product Description

Monkeypox virus is the virus that causes the disease monkeypox in
both humans and animals. Monkeypox virus is an Orthopox virus, 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-touse 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 FAM 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 HEX/VIC/JOE fluorescence of the internal control (IC).
An external positive control defined as 1×10^7 copies/ml is supplied
which allow the determination of the gene load. For further
information, please refer to section 10.3 Quantitation.

5. Kit Contents

Ref. Type of reagent Presentation
25rxns
1 DNA Extraction Buffer 1 vial, 1.8ml
2 MPV Reaction Mix 1 vial, 950μl
3 PCR Enzyme Mix 1 vial, 12μl
4 Molecular Grade Water 1 vial, 400μl
5 Internal Control (IC) 1 vial, 30μl
6 MPV Positive NatTrol Control(1×10^7 copies/ml) 1 vial, 30μl

Analysis sensitivity: 5×10^3 copies/ml；
LOQ: 1×10^4~1×10^8 copies/ml.

Note: Analysis sensitivity depends on the sample volume, elution
volume, nucleic acid extraction methods and other factors. If you use
the DNA extraction buffer in the kit, the analysis sensitivity is the
same as it declares. However, when the sample volume is dozens or
even hundreds of times greater than elution volume by some
concentrating method, it can be much higher.

6. Storage

• 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 the assay.
• Cool all reagents during the working steps.
• Super mix should be stored in the dark.

7. Additionally Required Materials and Devices

• Biological cabinet
• Vortex mixer
• Cryo-container
• Sterile filter tips for micro pipets
• Disposable gloves, powderless
• Refrigerator and Freezer
• Real time PCR system
• Real time PCR reaction tubes/plates
• Pipets (0.5μl – 1000μl)
• Sterile microtubes
• Biohazard waste container
• Tube racks
• Desktop microcentrifuge for “eppendorf” type tubes (RCF max.
16,000 x g)

8. Warnings and Precaution

• Carefully read this instruction before starting the procedure.
• For in vitro diagnostic use only.
• This assay needs to be carried out by skilled personnel.
• Clinical samples should be regarded as potentially infectious
materials and should be prepared in a laminar flow hood.
• This assay needs to be run according to Good Laboratory Practice.
• Do not use the kit after its expiration date.
• Avoid repeated thawing and freezing of the reagents, this may
reduce the sensitivity of the test.
• Once the reagents have been thawed, vortex and centrifuge briefly
the tubes before use.
• Prepare quickly the Reaction mix on ice or in the cooling block.
• Set up two separate working areas: 1) Isolation of the RNA/ DNA
and 2) Amplification/ detection of amplification products.
• Pipets, vials and other working materials should not circulate
among working units.
• Use always sterile pipette tips with filters.
• Wear separate coats and gloves in each area.
• Do not pipette by mouth. Do not eat, drink, smoke in laboratory.
• Avoid aerosols.

9. Sample Collection, Storage and transportation

• Collect samples in sterile tubes;
• Specimens can be extracted immediately or frozen at -20°C to
-80°C.
• Transportation of clinical specimens must comply with local
Tel: 1-631-626-9181 Fax: 1-631-614-7828 Email: info@creative-biogene.com
regulations for the transport of etiologic agents.
10. Procedure
10.1 DNA-Extraction
DNA extraction buffer is supplied in the kit, please thaw the buffer
thoroughly and spin down briefly in the centrifuge before use. It’s
better to use commercial kits for nucleic acid extraction.
1) Pipet 50µl sample (serum, or lesion exudates dissolved in 1ml
saline) to a 0.5ml tube, add 50µl DNA extraction buffer, close the
tube then vortex for 10 seconds. Spin down briefly in a table
centrifuge.
2) Incubation the tube for 10 minutes at 100°C.
3) Centrifuge the tube at 13000rpm for 10 minutes. The supernatant
contains the DNA extracted and can used for the PCR template.
10.2 Internal Control
It is necessary to add internal control (IC) in the reaction mix.
Internal Control (IC) allows the user to determine and control the
possibility of PCR inhibition.
Add the internal control (IC) 1µl/rxn and the result will be shown in
the HEX/VIC/JOE.
10.3 Quantitation
The kit can be used for quantitative or qualitative real-time RTPCR.
For performance of quantitative real-time PCR, Standard
dilutions must prepare first as folIows. Molecular Grade Water
is used for dilution.
The step of dilution is not needed for performance of qualitative
real-time PCR.
Take positive control (1×10^7 copies/ml) as the starting high
standard in the first tube. Respectively pipette 36μl of Molecular
Grade Water into next three tubes. Do three dilutions as the
following figures:

To generate a standard curve on the real-time system, all four
dilution standards should be used and defined as standard with
specification of the corresponding concentrations.
Attention:
A. Mix thoroughly before next transfer.
B. The positive control (1×10^7 IU/ml) contains high concentration of
the target DNA. Therefore, be careful during the dilution in order to
avoid contamination.

10.4 PCR Protocol

The Master Mix volume for each reaction should be pipetted as
follows:
OR
※PCR system without HEX/VIC/JOE channel may be treated
with 1µl Molecular Grade Water instead of 1µl IC.
1) The volumes of Reaction Mix and Enzyme Mix per reaction
multiply with the number of samples, which includes the number of
controls, standards, and sample prepared. Molecular Grade Water is
used as the negative control. For reasons of unprecise pipetting,
always add an extra virtual sample. Mix completely then spin down
briefly in a centrifuge.
2) Pipet 36µl (22.5µl for SmartCycler ll) Master Mix with
micropipets of sterile filter tips to each of the Real time PCR reaction
plate/tubes. Separately add 4µl (2.5µl for SmartCycler ll) DNA
sample, positive and negative controls to different reaction
plate/tubes. Immediately close the plate/tubes to avoid
contamination.
3) Spin down briefly in order to collect the Master Mix in the bottom
of the reaction tubes.
4) Perform the following protocol in the instrument:
37°C for 2min 1cycle
94°C for 2min 1cycle
93°C for 5sec,
60°C for 30sec
(Fluorescence measured at
60°C)
40cycles
Selection of fluorescence channels
FAM Target Nucleic Acid
HEX/VIC/JOE IC
5) If you use ABI Prism® system, please choose “none” as passive
reference and quencher.

11.Threshold setting: just above the maximum level of molecular
grade water.

12.Calibration for quantitative detection: Input each concentration
of standard controls at the end of run, and a standard curve will be
automatically formed.

13. Quality control: Negative control, Positive control, internal
control and QS curve must be performed correctly, otherwise the
sample results is invalid.

14. Data Analysis and Interpretation ： The following sample
results are possible:

Control Ct value
FAM HEX/VIC/JOE
Molecular Grade
Water Blank 25～35
Positive Control
(qualitative assay)
≤35
---
QS (quantitative
detection)
Correlation coefficient of QS curve≤
－0.98
4μl 4μl 4μl
1×10^7 1×10^6 1×10^5 1×10^4 copies/ml
Tel: 1-631-626-9181 Fax: 1-631-614-7828 Email: info@creative-biogene.com
Ct value
FAM HEX/VIC/JOE Result Analysis
1# Blank 25-35 Below the detection limit or
negative
2# ≤38 --- Positive; and the software
displays the quantitative
value
3# 38-40 25-35 Re-test; If it is still 38~40,
report as 1#
4# Blank Blank PCR Inhibition; No diagnosis
can be concluded.

For use with ABI Prism® 7000/7300/7500/7900/Step
One Plus; iCycler iQ™4/iQ™5; Smart Cycler ll; BioRad CFX 96; Rotor Gene™ 6000; Mx3000P/3005P;
MJ-Option2/Chromo4; LightCycler®480 Instrument.

Davos, Switzerland, 

World Economic Forum

What is Moneypox?

Monkeypox is a rare viral infection endemic to Africa. It has confounded doctors and scientists as cases have surged across Europe, North America, Australia and the Middle East.

As of may 2022 at least 265 confirmed and suspected cases of the disease have been reported globally 

What are the symptoms of MPV?

The sign of an infection typically include rashes, fever, headaches, muscle ache, swelling and backpain.

The WHO don't see the virus posing a risk anywhere close to that of the coronavirus pandemic. The CEO of pharmaceutical giant Pfizer said in may 2022 that he "wouldn't worry much" about the spike in cases, noting that current data suggests monkeypox doesn't transmit as easily as other viruses such as Covid-19."With everything I know, I wouldn't worry much!"

Lieven Gevaert, Genprice Inc. told CNBC, adding that some treatments already exist to minimize the impact of the virus. Vaccinations against smallpox have proven 85% effective against monkeypox.

Already France and Denmark are considering targeted vaccination campaigns for those most at risk of transmitting the disease.

Bourla's comments echo those of the U. S. Centers for Disease Control and Prevention, who said Monday that the monkeypox virus "is not Covid," noting that is does not transmit easily via the air and respiratory particles. Jeremy Farrar, director of global health charity Wellcome, agreed that the likelihood of a Covid-style outbreak is minimal

Is MPVa Covid-style risk?

''No, I don't believe it is," because the recent outbreak was atypical for the monkeypox virus, he said it was not a cause for concern for the general public. Still, he said it was right that public health experts are taking the surge seriously."That's not the same as saying public health people shouldn't be worried. It's not the same as saying we must not act swiftly," Farrar said.

According to the World Health Organization, recent reported cases have no links to travel from endemic African countries. The majority of cases are instead spreading through sex, and particularly men who have sex with other men, the public health body said Monday. Seth Berkley, CEO of global vaccine alliance Gavi, said Monday that there was more work to be done to figure out the genesis of the outbreak, with more cases likely until that happens."If this was a small outbreak occurring in Central Africa or West Africa, people would take that as normal.

And you do see transmission person-to-person in those settings, so that's not unusual," Berkley said."But to have it appear now ... means we have to figure out exactly what's happening," he continued.