JP-2025515495-A5 -

Dates

Publication Date

20260511

Application Date

20230427

Description

For the purposes of this disclosure, certain aspects, advantages, and novel features are described herein. Not all such advantages can necessarily be achieved according to any particular embodiment. Accordingly, a person skilled in the art will recognize that this disclosure can be embodied or implemented in a manner in which, for example, one advantage or group of advantages taught herein is achieved, while other advantages that can be taught or suggested herein are not necessarily achieved. Another aspect of the present invention may be as follows: [1] A method for analyzing a mixture suspected to contain multiple target nucleic acids, a) Processing a sample suspected to contain multiple different target nucleic acids in a pre-amplification reaction mixture to produce a multiplex pre-amplified mixture, The pre-amplification reaction mixture comprises at least four different target-specific primer pairs for generating amplification regions from at least four different target nucleic acids (if present in the sample), and at least one reference primer pair for generating amplification regions from a reference nucleic acid. Each of the primers in the pair of at least four different target-specific primers and the pair of at least one reference primers is at essentially the same concentration, produced by the process. b) Adding a portion of the pre-amplified multiplex mixture to the multiplex PCR assay reaction mixture, wherein the multiplex PCR assay reaction mixture is i) An additional amount of each of the at least four different target-specific primer pairs and the reference primer pair, wherein the primer in the additional amount of the at least four different target-specific primer pairs and the at least one reference primer pair is added in essentially the same amount, ii) at least four different target-specific probe oligonucleotides, each of which hybridizes specifically to one of the at least four different target nucleic acids (step a), and the at least four different target-specific probe oligonucleotides, each comprising a first label, and iii) A reference probe flap oligonucleotide that specifically hybridizes to the amplification region of the reference nucleic acid, comprising a second label, the addition of the reference probe flap oligonucleotide, Furthermore c) Performing a PCR assay using the multiplex PCR assay reaction mixture, wherein each of the reference nucleic acid region and the at least four different target regions (if amplified in step a) is amplified in the PCR assay reaction mixture, the target-specific probe oligonucleotide and the reference probe oligonucleotide specifically hybridize to the target region and the reference nucleic acid region amplified in the multiplex PCR assay reaction mixture, are cleaved to release the first and second labels, and the released first and second labels are measured. The method, including the method described above. [2] The method according to [1], wherein the pre-amplification reaction mixture includes a low-bias amplification buffer. [3] The method according to [2], wherein the multiplex PCR assay reaction mixture includes a low-bias amplification buffer, preferably the same low-bias amplification buffer used in the pre-amplification reaction mixture. [4] The method according to any one of [1] to [3], wherein in step b), the at least four different target-specific probe oligonucleotides and the reference probe flap oligonucleotide are present in essentially the same concentration in the multiplex PCR assay reaction mixture. [5] The method according to any one of [1] to [4], wherein the first label comprises a first 5' flap sequence, and the first 5' flap sequence is not substantially complementary to any of the amplification regions from the at least four different target nucleic acids. [6] The method according to [5], wherein the second label comprises a second 5' flap sequence, the second 5' flap sequence being different from the first 5' flap sequence and not substantially complementary to the amplification region from the reference nucleic acid. [7] The method according to [5] or [6], wherein the PCR assay reaction mixture further comprises a first FRET cassette labeled with a first fluorophore and/or a second FRET cassette labeled with a second fluorophore, the first FRET cassette comprising a sequence complementary to the first 5' flap sequence and the second FRET cassette comprising a sequence complementary to the second 5' flap sequence. [8] The method according to any one of [1] to [7], wherein the PCR assay reaction mixture further comprises a flap endonuclease, preferably a FEN-1 endonuclease, preferably an archaeal FEN-1 endonuclease. [9] The method according to any one of [1] to [8], wherein the first label comprises a first FRET system containing a first fluorophore, and the second label comprises a second FRET system containing a second fluorophore, and fluorescence from the first fluorophore and the second fluorophore is me