RayBio® Mpox Virus (MPXV) PCR Nucleic
Acid Detection Kit
Catalog #: PCR-MPXV
User Manual
Last revised: 10-09-2024
Caution:
Extraordinarily useful information enclosed
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RayBiotech, Inc.
RayBio® Mpox Virus (MPXV) PCR
Nucleic Acid Detection Kit Protocol
Please read the entire manual carefully before starting your
experiment
INTRODUCTION
Mpox virus is a virus from the orthopox family of viruses, a family most often associated with the
more severe smallpox virus (variola), or the origination of vaccinations from the cowpox virus.
Originally named for an outbreak among monkeys, it was first identified in humans in central and
western Africa around 1970 where it continues to circulate at a low endemic level. While the virus
is clinically less severe than smallpox, the symptoms are similar. With vaccinations against
smallpox ceasing in 1972 and the eradication of the smallpox virus, vaccinations in the younger
population (<50 years of age) is extremely limited. The escape from the endemic regions has the
World Health Organization on alert given the lack of associated travel within the region of those
suspected and confirmed cases. Understanding how the virus is being spread, the populations it
is spreading to, and the potential immune responses against the virus are of increasing
importance in the research field.
The RayBiotech Mpox Virus PCR Nucleic Acid Detection Kit is a ready to use PCR assay for the
detection of MPXV DNA in a liquid sample. It uses two sets of specific primers and probes at the
same fluorescent channel to detect the J2L and B6R genes of MPXV to enhance its sensitivity. A
set of primers specific to a conserved region of all orthopox viruses were also used to evaluate
possible false negative caused by virus genome mutation. Primer and probe for an internal control,
RNase P are also integrated in the assay to validate the assay quality.
PACKAGING SPECIFICATIONS
96 tests/box
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PURPOSE
This kit is used for the qualitative in vitro detection of Mpox virus (MPXV) nucleic acid from
purified DNA samples. Sample DNA should be purified according to manufacturer or related
procedures.
KIT COMPONENTS
Component Catalogue # Ingredients Specification Quantity
2x PCR
Buffer, dNTP’s, enzyme, ROX
Reaction PCR-MPXV-MM 1000µL / tube 1 tube
reference dye
Solution
MPXVJ2L, B6R/Orthopox
Primers and
E9L/RNase P Primer & Probe 500µL / tube 1 tube
Probe Mix PCR-MPXV-PP
Mix
Positive Synthetic MPXV genomic
50µL / tube 1 tube
Control PCR-POS-POS DNA/RNase P Positive Control
Negative
Nuclease-free water 500µL / tube 1 tube
Control PCR-MPXV-NEG
Note: Do not mix reagents from different lots.
Note: PCR machines may require specific PCR plate types. Please refer to the manufacturer’s
recommendation for PCR plates before running the assay on your PCR machine.
STORAGE AND EXPIRATION
The kit can be stored at -20°C for a period of 12 months prior to opening. After opening, the
reagents are valid for at least 6 months if stored at -20°C.
REQUIRED MATERIALS (NOT INCLUDED)
• Sample DNA purification: can be any common commercially available genomic DNA
purifications kits
• DNA preservation solution
• Fluorescence PCR instrument capable of reading FAM or equivalent channel (494 nm
maximum absorption, 518 nm maximum emission), Cy5 channel (640 nm maximum
absorption, 682 nm maximum emission) and TAMRA or equivalent channel (550 nm
maximum absorption, 586 nm maximum emission).
• Vortex Mixer
• Microcentrifuge
• Pipettes
• Sterile nuclease-free pipette tips (barrier tips recommended) and microfuge tubes
• Compatible PCR Plate
• Contact our technical support team for questions about compatibility:
techsupport@raybiotech.com
SAMPLE REQUIREMENTS
1. This is a Research Use Only (RUO) kit and is not to be used for diagnostic purposes of any
kind.
2. Sample types: Samples should be purified DNA using commonly available lab practices like
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Trizol related methods, or commercially available DNA purification kits. Please follow
manufacturer’s guidelines with respect to any sample purification steps. The final sample DNA
amount added to the assay should not exceed 100 ng.
3. All specimens, regardless of how or for what purpose they were collected, should be regarded
as potentially infectious and handled with extreme caution. Measures should be taken to
minimize the risk of laboratory transmission based on risk assessment when testing any
samples. These precautions, at a minimum, should include proficiency and competency
testing, appropriate PPE, avoiding aerosols, and using effective disinfectants (quaternary
ammonium compounds and 0.5% bleach).
GENERAL CONSIDERATIONS
1. To prevent contamination of PCR reactions, clean and decontaminate all working surfaces,
centrifuges, pipets, and other equipment with 10% bleach or DNA Away®, followed by 70%
Ethanol before every assay.
2. Conduct sample processing and DNA extraction in a separate area (below termed the
“Sample Processing Area”) from the PCR assay setup (below termed the “PCR Assay
Setup Area”).
3. Care should be taken to avoid contamination of samples and reactions with DNA amplicons
from used PCR plates. It is thus recommended to place used PCR plates in a sealed bag
and discard them appropriately. Never open a used PCR plate or place it in the same area
as the PCR instrument.
4. To minimize cross-contamination between experimental samples, disposable pipettes and
filtered pipette tips are recommended.
TESTING METHOD
1. Sample Processing (Sample Processing Area)
1.1. Sample Inactivation and Preservation: Use a DNA sample preservation solution for
virus inactivation and DNA preservation.
1.2. Sample Extraction and Purification: Common commercial nucleic acid extraction and
purification kits, or Trizol-based extraction method, may be used to extract the nucleic
acid.
2. Assay Assembly (PCR Assay Setup Area)
2.1 Thaw reagents: Remove all components from the kit, and fully thaw to room
temperature. After thawing, mix gently by pipetting. Briefly centrifuge to collect contents at
bottom of vial.
2.2 Calculate number of reactions needed: The number of reactions to be prepared per
PCR run maybe calculated by (# of singly run samples to be tested + 2). Adding 2 to the
number of samples to be tested takes positive and negative controls tests into account. It is
recommended to include 1 positive and 1 negative control with each experiment. Refer to
Table 1 for a summary of reaction components included in each well. NOTE: it is
recommended to make 1 or 2 additional reaction volumes to account for pipetting error.
2.3 Prepare PCR Master Mix: As outlined in Table 1, each reaction should contain 10µL 2x
PCR Reaction Solution and 5 µL Primers and Probe Mix. To calculate the total volume
necessary for the run, multiply the volumes of each component by the number of reactions
calculated in 2.2 above. Mix the PCR Reaction Solution and other components together to
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prepare a Master Mix.
Table 1: Reaction Components for Samples and Controls.
Positive Control Negative Control
Component Sample Reaction
Reaction Reaction
2xPCR Reaction
10 µl 10 µl 10 µl
Solution
Primers and
5 µl 5 µl 5 µl
Probe Mix
Positive Control 5 µl -- --
Negative Control -- 5 µl --
Sample -- -- 5 µl
Total Volume 20µl 20µl 20µl
3. Sample Loading (PCR Assay Setup Area)
3.1 Add 15 µl of prepared PCR Master Mix from 2.3 above to each well of a PCR reaction
plate.
3.2 Add 5 µl of sample template DNA (no more than 100ng recommended) to each well
and pipette up and down at least 5 times to mix.
3.3 Add at least 1 positive control and 1 negative control samples.
3.4 Seal the plate or tubes tightly.
3.5 Centrifuge the plate or tubes for 30 seconds at low speed. Note: The sealed PCR
reaction tubes can be stored at 2-8°C for up to 4 hours before the “PCR Amplification”
step 4 below.
4. PCR Amplification (PCR Assay Setup Area)
4.1 Sample setup: Set the sample number, targets, negative control, and positive control
accordingly to your plate setup.
4.2 Fluorescence Channel Selection: Select FAM (or equivalent channel) and set the target
name for “MPXV”, this channel will detect both J2L and B6R viral gene. Select TAMRA (or
equivalent channel) and set the target name for “Internal Control”, this channel will detect
the RNase P gene. Select Cy5 (or equivalent channel) and set the target name for
“Orthopox” E9L gene. This kit contains a reference fluorescence dye ROX (passive
reference) that will help decrease variation. In the PCR instrument setting, set the
reference dye to “ROX”.
4.3 Set reaction conditions according to Table 2. Set the reaction volume to 20 µl.
Table 2: PCR Program
Temperature Temperature Number of
Step Time
(°C) Ramp Rate Cycles
Stage 1 Initial Denaturation 95 1 min 1.6°C/sec 1
Denature 95 15 s 1.6°C/sec
Stage 2 Anneal, extend, detect 40
60 30 s 1.6°C/sec
fluorescence
4.4 Save the file and run program. A sample image of PCR amplification is shown (Figure1).
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5. Result and Analysis
The positive and negative control PCR reactions are considered valid if the negative and
positive controls meet the criteria listed in Table 3. The PCR reaction is invalid if 1) the
positive control does not have logarithmic growth or the Ct ≥ 36 or 2) the negative control
has a Ct ˂ 36. If the reaction is invalid, the measurement of all samples in this experiment
should be discarded, and the assay repeated.
Note: Since RNase P signal on TAMRA channel has lower background than Mpox signal on
FAM channel and Orthopox signal on Cy5 channel, researchers can present the RNase P
separately.
Table 3. Validation of PCR reactions with quality controls
Target Positive Negative Control
Control
MPXV Ct ≥ 36 or no
Ct ˂ 36
B6R/J2L Amplification
Orthopox Ct ≥ 36 or no
Ct ˂ 36
E9L Amplification
Ct ≥ 36 or no
RNase P Ct ˂ 36
Amplification
INTERPRETATION OF TEST RESULTS
The PCR reaction results are explained according to Tables 4 and 5.
Table 4. Interpretation of Individual PCR Reactions
PCR reaction results MPXV Ct RNase P
+ ˂ 36 ˂ 36
- No amplification, or Ct ≥ 36 No amplification, or Ct ≥ 36
Table 5. Interpretation of Sample Test
FAM (MPXV Cy5 (Orthopox TAMRA (PCR PCR Result
detection) virus detection) validation)
+ + + MPXV +
+ + - MPXV +
- - + MPXV -
- + + Retest needed
- - - Invalid PCR
Positive Result: the sample contains the target genes.
Negative Result: the sample does not contain the target genes.
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Invalid Result: the sample should be rerun with fresh samples and controls.
PRODUCT PERFORMANCE INDEX
1. Limit of Detection: The LOD of the assay is 2 copies/µL, or 10 copies per reaction (5µL of
sample volume).
2. Repeatability: Precision testing showed that the coefficient of variation of the
precision Ct values within this kit lot are ≤ 1.2%. Repeatability between lots of product are to
be ≤ 10%.
Specificity
No cross reactivity was identified when the kit was evaluated against other common Orthopox
family members: Cowpox virus, Camelpox virus, and Vaccinia virus.
Figure 1. Five microliter of positive control contains 102 copies/µl of Mpox Virus B6R and J2L
Synthetic DNA (Grey color, FAM channel), 102 copies/µl of Orthopox virus J2L Synthetic DNA
(Yellow color, Cy5 channel) and fixed amount of RNase P template (Green color, TAMRA
channel) were added to 15ul of master mix and amplified in a QuantStudio™ 5 Real-Time PCR.
