Sequence detection assay

摘要

There is provided a method of detecting the presence in a sample of a first target sequence and a second target sequence within a test region of a nucleic acid sequence comprising conducting a nucleic acid amplification reaction, to form a forward amplicon strand and a reverse amplicon strand of the test region, contacting the forward amplicon strand with a first probe labelled with a first FRET label and capable of hybridising to the first target sequence of complement thereof in the forward amplicon strand, and contacting the reverse amplicon strand with a second probe labelled with a second FRET label and capable of hybridising to the second target sequence or complement thereof in the reverse amplicon strand; wherein the nucleic acid amplification reaction is conducted using a forward amplification primer labelled with a third FRET label and a reverse amplification primer labelled with a fourth FRET label, the forward primer being incorporated into the forward amplicon strand and the second primer being incorporated into the reverse amplicon strand during the amplification reaction; and further wherein the first and third FRET labels form a FRET pair and the second and fourth FRET labels form a different FRET pair, each FRET pair comprising a donor label; the method further comprising the steps of exposing the sample to an excitation source having a wavelength which excites the donor label in the first FRET pair and the donor label in the second FRET pair, detecting fluorescence from the sample and relating this to the presence or absence of the first and second target sequences.