Taylor-Leigh Siebritz
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Real-time PCR (Polymerase Chain Reaction)
Real-time PCR (Polymerase Chain Reaction)
The use of PCR techniques identifies the Giardia lamblia genetic material, and confirms an infection with high species-specificity (Vicente, et al., 2024).
Steps in G. lamblia PCR:
Steps in G. lamblia PCR:
DNA isolation:
To optomise cyst disruption, the faecal sample is subjected to two cycles of freezing and thawing (in liquid nitrogen and a warm water bath at 70C for 5 minutes each); followed by a repeat cycle of another 5 minutes in liquid nitrogen, and warm water at 95C (Coradi, et al., 2011). DNA is then extracted, using DNA stool mini kits and the prescribed protocol of elusion in 100ul and storage at a temperature of 80C. The QIAamp mini kit is often used (Alharbi, et al., 2020).
Real-time PCR amplification:
The SSU rRNA gene of G. lamblia is targeted by the Giardia-specific primers: the forward primer Giardia-F and the reverse primer, Giardia-R, as well as the Giardia-specific double-labeled probe Giardia-T (Alharbi, et al., 2020).
PCR Assay:
With two loci - glutamate dehydrogenase (gdh) and triose phosphate isomerase (tpi) genes - eluted DNA is amplified, with a nested PCR reaction. The PCR products are then visualised using 1.5% agarose gel electrophoresis, stained with ethidium bromide and transilluminator UV light (Coradi, et al., 2011).
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