Monkeypox Virus Real Time PCR Kit

1. Sample Collection: Specimen should be collected into sterile tubes in accordance

with standard technical specifications.

2. Reagent Preparation (Reagent Preparation Area)

Take out the components of the kit, balance them at room temperature for standby use.

3. Specimen Processing (Specimen Processing Area)

3.1 Nucleic acid extraction

It is recommended to take 200μL liquid samples, Positive Control and Negative Control for nucleic acid extraction, according to the corresponding requirements and procedures of viral DNA extraction kits.

3.2 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 should be treated as two samples.

(1) 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.

(2) Cover PCR tubes tightly, flick PCR tubes by hand until lyophilized powder is completely dissolved and blended, collect the liquid to the bottom of PCR tubes by instantaneous low-speed centrifugation.

(3) 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 10 μL 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.

4. PCR Amplification (Detection Area)

4.1 Put the PCR tubes/reaction chip into the reaction tank and set the names of each reaction well in the corresponding order.

4.2 Settings of detection fluorescence: (1) Monkeypox virus (FAM); (2) Internal Control (CY5).

4.3 Run the following cycling protocol

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

 Protocol of BTK-8:

5. 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 result column. The negative and positive control results shall conform to the following "6. Quality Control ".

6. Quality Control

6.1 Negative Control: No Ct or Ct>40 in FAM channel, Ct≤40 in CY5 channel with normal amplification curve.

6.2 Positive Control: Ct≤35 in FAM channel with normal amplification curve, Ct≤40 in CY5 channel with normal amplification curve.

6.3 The result is valid if all the above criteria is met. Otherwise, the result is invalid.

Result Interpretation

The following results are possible:

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