Probe system for real-time quantitative and qualitative analysis of biomaterial, reaction chamber with said probe system, and analysis method thereof
Inventors
Kim, Lee Kyung • Paek, Mun Cheol • Ku, Su Jin • PARK, Sun Young • Park, Jong Pil • Lee, Do Bu • Kim, Nam Joong • Noh, Jin Seok
Assignees
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Publication Number
US-10428372-B2
Publication Date
2019-10-01
Expiration Date
Abstract
A probe system for real-time quantitative and qualitative analysis of a biomaterial, and a reaction chamber with the probe system, and an analysis method thereof are provided. The probe system, which is included in the reaction chamber having an optically transmissive flat bottom surface and having a test sample accommodated therein, includes a target probe-reporter probe linker accommodated in the reaction chamber and including a target probe, which includes a sequence complementary to a target nucleic acid sequence to be detected, a first fluorophore and a first quencher, and a reporter probe linked to an end of the target probe and including a sequence non-complementary to the target nucleic acid sequence, and a capture probe included in a biochip formed on a bottom surface of the reaction chamber and including a complementary sequence hybridizable with the non-complementary sequence of the reporter probe, a second fluorophore and a second quencher.
Core Innovation
The invention provides a probe system and a hermetically closed reaction chamber for real-time quantitative and qualitative analysis of a biomaterial. A test sample is introduced into a reaction chamber having a top opening and is hermetically closed with a cap after introduction, and a bottom surface of the reaction chamber comprises an optically transmissive window having an optically transmissive flat surface, with a biochip formed on the optically transmissive window for in situ fluorescence analysis.
A target probe-reporter probe linker is accommodated in the reaction chamber, wherein the target probe has a sequence complementary to a target nucleic acid sequence to be detected and includes a first fluorophore and a first quencher. A reporter probe is linked to an end of the target probe and has a sequence non-complementary to the target nucleic acid sequence, and when the target probe is hybridized with target nucleic acids, a first fluorescence signal is generated while the target probe is digested with a polymerase having nuclease activities, and the reporter probe is digested from the target probe and released into the reaction chamber.
The biochip comprises a capture probe having a complementary sequence capable of hybridizing with the non-complementary sequence of the released reporter probe, and the capture probe comprises a second fluorophore and a second quencher. Upon hybridizing, a second fluorescence signal is emitted due to a structural change of the capture probe caused by an extension reaction, and quantitative analysis is performed from the first fluorescence signal while qualitative analysis is performed from the second fluorescence signal in real time, with different wavelength detection optionally used for the first and second fluorescence signals.
Claims Coverage
The independent claim is directed to a method for real-time quantitative and qualitative analysis of target nucleic acids in a biomaterial using a hermetically closed reaction chamber with an optically transmissive window and a biochip. It includes three main inventive aspects: the structure and placement of a target probe-reporter probe linker with a fluorescence pair, sequential digestion and release followed by hybridization on the biochip producing a second fluorescence signal via an extension-induced structural change, and detecting both fluorescence signals for quantitative and qualitative analysis.
Hermetically closed reaction chamber with optically transmissive window and in situ biochip
The reaction chamber has a top opening, is hermetically closed with a cap after introduction of the test sample, and has a bottom surface comprising an optically transmissive window with an optically transmissive flat surface, where a biochip is formed on the optically transmissive window.
Target probe-reporter probe linker for digestion-driven release and first fluorescence signal
A target probe-reporter probe linker comprises a target probe complementary to a target nucleic acid sequence and including a first fluorophore and a first quencher, and a reporter probe linked to an end of the target probe having a sequence non-complementary to the target nucleic acid sequence; the method uses polymerase having nuclease activities to digest the target probe and digest the reporter probe from the target probe to release the reporter probe into the reaction chamber while generating a first fluorescence signal from the first fluorophore.
Biochip capture probe for hybridization with released reporter probe and extension-induced second fluorescence signal
The biochip includes a capture probe with a complementary sequence capable of hybridizing with the non-complementary sequence of the reporter probe and including a second fluorophore and a second quencher, where after hybridization with the released reporter probe the capture probe emits a second fluorescence signal due to a structural change of the capture probe caused by an extension reaction.
Real-time detection and assignment of quantitative and qualitative analysis
The method detects the first and second fluorescence signals, performing quantitative analysis of the target nucleic acids from the first fluorescence signal and performing qualitative analysis of the target nucleic acids from the second fluorescence signal.
The core coverage is a real-time dual-signal approach in which polymerase nuclease activities generate a first fluorescence signal and release a reporter probe that is captured on a biochip to generate a second fluorescence signal via an extension-induced structural change; quantitative analysis is assigned to the first fluorescence signal and qualitative analysis to the second fluorescence signal.
Stated Advantages
Enables real-time quantitative analysis of target nucleic acids from the first fluorescence signal.
Enables real-time qualitative analysis of target nucleic acids from the second fluorescence signal.
Documented Applications
An illustrative Mycobacterium assay example is described, including Mycobacterium tuberculosis and nontuberculous Mycobacterium with strain quantification and genotype discrimination using different fluorophore sets.
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