New method of detection of several target nucleic acids in a biological sample

EP4728095A1Pending Publication Date: 2026-04-22INNOVATIVE DIAGNOSTICS
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Patent Information

Authority / Receiving Office
EP · EP
Patent Type
Applications
Current Assignee / Owner
INNOVATIVE DIAGNOSTICS
Filing Date
2024-06-11
Publication Date
2026-04-22

AI Technical Summary

Technical Problem

Current nucleic acid detection methods are limited in their ability to simultaneously detect multiple target sequences from a single biological sample due to sensitivity to non-specific signals and genetic variations, and they often require multiple fluorescence channels, which increases complexity and analysis time.

Method used

The method involves using a tag sequence linked to the 5’-end of primers and a specific probe sequence with a 3’ blocker to hybridize with the extended neosynthesized single-strand, allowing for the detection of multiple target nucleic acid sequences on the same fluorescence channel without interference from genetic variations, and can be combined with real-time PCR using TaqMan probes for increased multiplexing capacity.

Benefits of technology

This approach enables the independent detection of multiple target sequences without interpretation errors due to genetic variations, reduces analysis time, and increases multiplexing capacity, allowing for the simultaneous detection of up to 25 targets in a single reaction mix, while maintaining specificity and sensitivity.

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Abstract

The present invention concerns an in vitro method of detecting at least two target nucleic acid sequences from a biological sample comprising: a) releasing and / or extracting the nucleic acids from the biological sample, and b) amplifying and detecting at least two target nucleic acid sequences in a PCR reaction using the nucleic acids of step a) as a template, wherein the DNA amplification and detection step b) comprises: (b1) hybridizing each target nucleic acid sequence with a specific primers pair composed of a forward primer and a reverse primer comprising nucleotide sequences complementary to the target nucleic acid sequence, wherein at least one primer of the pair additionally comprises a tag sequence that is linked to its 5'-end portion; (b2) contacting the resultant of step b1) with a template-dependent nucleic acid polymerase under primer extension conditions for inducing the 3'-extension reaction and forming an extended neosynthetized strand; (b3) denaturing the amplified product of step (b2) and contacting the extended neosynthetized single-strand with a probe sequence comprising a 3' blocker and an interactive label, wherein the probe sequence hybridizes with the 3'-end portion of the extended neosynthetized single-strand and forms, under conditions for inducing the 3'-extension reaction, an extended duplex providing the signal; and c) optionally quantifying the amount of the said target nucleic acids.
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