A fluorescent based PCR assay for the detection and quantitation of Giardia duodenalis genotypes in mixed populations

Abstract

A method based on the polymerase chain reaction has been developed for differentiating between genotypically and phenotypically distinct strains of Giardia duodenalis and quantifying the amount of initial template of the different genotypes in mixed populations. The assay relies on a sequence-specific probe, labelled with two fluorescent dyes, designed to bind within the small subunit ribosomal (SSU) RNA gene. This target region is amplified by primers specific for either Group 1 or Group 2-type isolates of G. duodenalis and the probe binds within the primer-targeted region. This quantitative method takes advantage of the 5′ nuclease activity of Taq DNA polymerase, which, on encountering a probe bound within the target DNA sequence cleaves it, causing it to become dissociated from the template. When the two fluorescent dyes bound to the probe are in close proximity (when the probe is intact), the interaction of the two dyes prevents the reporter dye from fluorescing. However, during the extension phase of amplification, the activity of the DNA polymerase causes the dyes to become separated and hence the reporter dye increases its fluorescent intensity. This release of fluorescence is directly related to the amplified amount of target template. This assay was developed with the aim of providing a unique method with which to investigate interactions within mixed populations of genetically distinct strains of G. duodenalis.