Concept of Polymerase Chain Reaction [PCR] Amplification of the Target

GeneXpert MTB/RIF assay targets rpoB gene that encodes for RNA polymerase β subunit.

• More than 95% of mutations in the rpoB gene are located in the 81 base pair core regions (codons 507 – 533) also known as Rifampicin Resistance Determining Region (RRDR).
• Mutations lead to inhibition of binding of rifampicin to RNA polymerase. This results in the development of RIF resistance by the mycobacteria.
• Semi-nested polymerase chain reaction (PCR) using a set of three primers P1, P2 and P3 for amplification of 192 bp of rpoB gene containing 81 bp RRDR
• 1^st round of amplification takes place using primers P1 and P3 (19 cycles)
• In the next round, P2 and P3 are used to amplify the target (40 cycles)

Resources

• GeneXpert package insert

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