Monkeypox Virus nucleic acid detection kit (Fluorescence PCR method)

Cat. No.: BT-PR2023-D


* Lead Time: 10 days
* Not FDA Approved
* Professional Lab Only
* Ask for your inquiry of more than 2 orders


SPECIFICATION:  BT-PR2023-D: 48 tests/kit


INTENDED USE
This kit is used to achieve qualitative detection of Monkeypox viral DNA extracted from
serum or lesion exudate samples of suspected infected patients and other patients requiring
diagnosis.

PRINCIPLE
This product is a fluorescent probe-based Taqman® real-time PCR assay system.
Specific primers and probes are designed for the detection of F3L gene of Monkeypox Virus. Internal control targeting the human conserved gene monitor the sample collection, sample handling and real-time PCR process to avoid false-negative results. The kit is a fully premix lyophilized system, which includes materials needed for Monkeypox Virus detection: nucleic acid amplification enzyme,UDG enzyme, reaction buffer, specific primer and probe.

KIT COMPONENTS
 

Components	Package	Ingredient
Monkeypox Virus Lyophilized premix	8 strip PCR tubes × 6 pouches	Primers, probes, dNTP/dUTP Mix, Mg2+, Taq DNA polymerase, UDG Enzyme
MPV Positive Control	400 μL×1 tube	DNA sequences containing Target gene
MPV Negative Control	400 μL×1 tube	DNA sequences containing human gene segment
Dissolving Solution	1 mL×1 tube	Stabilizer

Note:
1. Do not mix the components from different batches for detection.
2. Prepare nucleic acid release reagents by users. Recommend Nucleic Acid Extraction Reagent manufactured by Bioteke.
3. When using the same batch of reagents, negative and positive controls should be tested at least once in one day to ensure that the environment is contamination-free and the reagent & instrument are effective.
4. The reagent “Monkeypox Virus Lyophilized premix” should be used up in two hours after dissolution. Unpacked but unused “Lyophilized RT-PCR premix” can be sealed and stored at -20±5°C. If air leakage or deliquescence occurs, do not continue to use.


STORAGE & SHELF LIFE
The kit can be transported at room temperature (no more than one month). It can be stored at -20±5 °C for one year. Temporarily store at 4°C for no more than 6 months. Repeated freezing and thawing should not exceed 7 times.


INSTRUMENTS
Real-time PCR instrument such as ABI7500, Bio-Rad CFX96, SLAN-96S,QuantStudio and BTK-8.


SAMPLING & HANDLING
1. Suitable specimen type: serum,lesion exudate samples.
2. Collection: Specimen should be collected into sterile tubes in accordance with standard
technical specifications. Storage: The collected specimen should be used for detection immediately or frozen at ≤-20°C for no more than one month.Repeated freezing and thawing should be avoid.
3. Transport: The specimen should be transported using sealed foam box with ice.


PROTOCOL
1. Reagent Preparation (Reagent Preparation Area)
Take out the components of the kit, balance them at room temperature for standby use.
2. Specimen Processing (Specimen Processing Area)
2.1 Nucleic acid extraction
It is recommended to take 200 μL liquid samples, Positive Control and Negative Control for nucleic acid extraction, according to the corresponding requirements and procedures of viral DNA extraction kits.
2.2 Lyophilized powder dissolving and template addition
Prepare Monkeypox Virus Lyophilized premix according to the number of samples. One sample needs one PCR tube containing Lyophilized premix powder. Negative control & positive control are treated as two samples.

First, add 15μL Dissolving Solution into each PCR tube containing Lyophilized premix, then add 5μL extracted samples/Negative Control/Positive Control into each PCR tube respectively. Then cover PCR tubes tightly, flick PCR tubes by hand until lyophilized powder is completely dissolved and blended, and collect the liquid to the bottom of PCR tubes by instantaneous low-speed centrifugation.

If use common Real-time PCR instrument for detection, then directly transfer PCR tubes to the amplification area; if use BTK-8 for detection, then need to perform the following operations: Transfer 10μL liquid from PCR tube to the reaction chip well of BTK-8. One PCR tube corresponds to one well on the chip. During pipetting operation, ensure that the pipette is 90 degrees vertical. The aerosol barrier pipette tips should be placed in the center of the well with moderate force and stop pushing the pipette when it reaches the first gear (to avoid bubbles). After the wells are filled, take out a reaction chip membrane to cover all wells and the chip is then transferred to the amplification detection area.

3. PCR Amplification (Detection Area)
3.1 Put the PCR tubes/reaction chip into the reaction tank and set the names of each reaction
well in the corresponding order.
3.2 Settings of detection fluorescence: (1) Monkeypox virus (FAM); (2)Internal Control
(CY5).
3.3 Run the following cycling protocol:

Protocol of ABI7500, Bio-Rad CFX96, SLAN-96S,QuantStudio:

Steps		Temperature	Time	Cycles
1	Pre-denaturation	95 °C	2 min	1
2	Denaturation	95 °C	10 s	45
	Annealing, extension, fluorescence acquisition	60 °C	30 s

Protocol of BTK-8:

Steps		Temperature	Time	Cycles
1	Pre-denaturation	95 °C	1 min	1
2	Denaturation	95 °C	5 s	45
	Annealing, extension, fluorescence acquisition	60 °C	12 s

4. Results analysis (please refer to Instrument User Manual)
After the reaction, the results will be saved automatically. Click “Analyze” to analyze, and the instrument will automatically interpret Ct values of each sample in the result column. The negative and positive control results shall conform to the following "5. Quality
Control ".

5. QUALITY CONTROL
5.1 Negative Control: No Ct or Ct>40 in FAM channel, Ct≤40 in CY5 channel with normal amplification curve.
5.2 Positive Control: Ct≤35 in FAM channel with normal amplification curve, Ct≤40 in CY5 channel with normal amplification curve.
5.3 The result is valid if all the above criteria is met. Otherwise, the result is invalid.

TEST RESULTS INTERPRETATION
The following results are possible:

#	Ct value of FAM channel	Ct value of CY5 channel	Interpretation
1#	No Ct or Ct>40	≤40	Monkeypox virus negative
2#	≤40	Any result	Monkeypox virus positive
3#	40~45	≤40	Re-test; if it is still 40~45, report as 1#
4#	No Ct or Ct>40	No Ct or Ct>40	Invalid

NOTE: If invalid result occur, the sample needs to be collected and tested again.


ASSAY LIMITATIONS
1. This kit is only for scientific research, and it should not be directly used as theevidencefor clinical diagnosis and treatment. The clinical management of patients shouldbeconsidered in combination with their symptoms, medical history and exposure history.
2. The detection result can be affected by operations, including specimen collection, storageand transportation. False negative result may occur if there is any mistakes intheoperation.Cross contamination during specimen treatment may lead to false positive result.
3. The detected target sequences of this kit are the conserved region of Monkeypoxvirus’sF3L gene. However, target sequence variations may lead to false negative result.


PERFORMANCE SPECIFICATIONS
1. Detection limitation: 500 copies /mL.
2. Precision: The coefficient of variation (CV, %) of Ct value of within-batch/between-batch precision is ≤5%.
3. Accuracy: The conformity rate of negative/positive reference: 100%.
4. Specificity: no cross-reactivity with human genome and the following pathogens: smallpox virus, sheep pox virus, vaccinia virus, chicken pox virus,hepatitis B virus, hepatitis C virus, human immunodeficiency virus,enterovirus.


ATTENTIONS
1. This product is only used for scientific research.
2. The temperature should not exceed 37℃ when transporting the kit. If the outdoor temperature exceeds this temperature, please add ice packs during the transportation of the kit to control the transportation temperature below 37℃.
3. Use sterile, DNase-free and RNase-free tubes and tips during the detection.
4. During the detection, it is necessary to avoid cross-contamination, wear appropriate protective equipments, disposable gloves and masks, and complete the operation in the biosafety cabinet to avoid harmful substances entering the respiratory tract.
5. In case of contact with skin and mucous membrane during use, please rinse immediately with flowing water, which will not cause any harm to the operator.
6. All liquid reagents should be fully melted and mixed at room temperature before use, and centrifuged at 8,000 rpm for a few seconds before use.
7. After use, the packaging and waste liquid must be uniformly treated as medical waste to prevent pollution.


REFERENCES
[1] Kulesh D A,Loveless B M,Norwood D,et al. Monkeypox virus detection in rodents using real-time 3'-minor groove binder TaqMan assays on the Roche LightCycler.[J]. Laboratory Investigation, 2004, 84(9):1200-1208.
[2] Kurt,D,Reed,et al. The Detection of Monkeypox in Humans in the Western Hemisphere[J]. New England Journal of Medicine, 2004, 350(4).

 

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