Duplex one-step real-time RT-PCR assays

 

After RNA extraction, the test first relies on the reverse transcription (RT) of the RNA of FMD virus into complementary DNA (cDNA). The cDNA then undergoes PCR amplification by a DNA polymerase that uses primers and a TaqMan® probe, labelled with a FAM or VIC fluorochrome at the 5' end. The two enzymatic reactions are performed successively in the same tube (one-step, real-time RT-PCR).

Due to the high level of mutations generated during the replication of RNA viruses, it is recommended to use two specific primer pairs in parallel targeting two different regions of the FMD genome: the IRES region and the encoding region for the RNA-dependent RNA polymerase 3D.

In addition, a third pair of primers is used to detect the endogenous ß-actin cellular gene (present in the extracted cellular RNA fraction). Detection of this gene acts as a control for the extraction step and absence of PCR inhibitors.

This is a duplex reaction. In a single reaction, two targets are detected simultaneously: either the IRES + ß-actin targets, or the 3D + ß-actin targets. The probe for the FMD target is labelled with a FAM fluorochrome and the probe for the ß-actin target with a VIC fluorochrome.

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