MPXV PCR Monkeypox Virus Freeze-Dried Multiplex Real-Time PCR Detection

OPERATION PROCEDURE

1) Nucleic Acid Extraction Extract DNA from specimens using a proper extraction kit. It is recommended that the nucleic acid is eluted with about 100 μl of elution buffer (TE or nuclease-free H2O) in the final step of extraction. The purified nucleic acid should be used immediately or stored at -20°C.

2) Positive Control Preparation The positive control can be stored at ambient temperature before rehydration for up to 12 months. It should be rehydrated before use by adding 250 μl of TE buffer or nuclease-free H2O, vortexed at low speed for 15-20 seconds, and centrifuged at low speed for 15-20 seconds. Use immediately or store at -20°C.

3) Real-Time RT-PCR Mix Preparation

A) Open the vacuum package and take out the 8-tube strips containing the reagent. Check the pellet is at the bottom of the tube (Cut the number of tubes as needed if necessary). If the provided tubes are not compatible with your instrument, transfer the reagent pellet to an optical tube compatible with your instrument.

B) Open the tubes and discard the cap(s) (not suitable for real-time PCR machines), and prepare the reaction mix on ice as below

* Nuclease-free water can be used as negative control.

C) Seal the tubes with optical caps or 96-well plate with optical seal film (not provided).

D) Vortex the tubes at low speed for 10~15sec, and centrifuge at 3000rpm for 20sec and place them in a real-time PCR instrument.

3) RT-PCR Setup: Set the reaction volume as 25μl, and PCR amplification procedure as below. Collect FAM (for N gene), HEX (for E gene), ROX (for Orf1ab) and CY5 (for RNase P gene) fluorescence at 60°C, and select NONE as passive reference.
4) Result Analysis and Interpretation Set the threshold line according to instructions of the instrument.

A) Quality Control:

B) Sample Result Interpretation

Technical Parameters: