Method for detecting target nucleic acid sequence amplified by multiplex amplification double signal

Abstract

The present invention relates to a detection method (multiple amplification nested signal amplification; MANSA) of a target nucleic acid sequence amplified by multiple amplification double signals, in particular to a method for detecting a target nucleic acid sequence by using a dumbbell-shaped oligonucleotide for the first time A method for amplifying and detecting a target nucleic acid sequence through the amplification reaction and the second signal amplification reaction. The present invention can detect multiple target nucleic acid sequences in very limited samples without false positives.

Description

technical field [0001] The present invention relates to a detection method (multiple amplification nested signal amplification; MANSA) of a target nucleic acid sequence amplified by multiple amplification double signals, in particular to a method for detecting a target nucleic acid sequence by using a dumbbell-shaped oligonucleotide for the first time A method for amplifying and detecting a target nucleic acid sequence through the amplification reaction and the second signal amplification reaction. Background technique [0002] At present, the most common method used by researchers to obtain genetic samples is the polymerase chain reaction method using DNA polymerase. The oligonucleotides used in the polymerase chain reaction are designed to bind to opposite strands of the template DNA. Although the above method has the advantage of being able to accurately amplify only the desired fragment in the target gene by arbitrarily adjusting the length and base sequence of the olig...

AI Technical Summary

Problems solved by technology

Although the existing real-time PCR method has the advantage of being able to simultaneously perform amplification and detection in a homogeneous assay, the number of target nucleic acid sequences that can be detected simultaneously will also be limited because the types of fluorescent indicator molecules are limited. restricted

The existing thermal cycler (thermocycler) that can detect target nucleic acid sequences in real time can detect 5-plex at most at the same time, so the number of target nucleic acid sequences that can be detected at the same time will also be limited, and in order to test large-capacity It takes a lot of time to analyze the raw materials, and it needs to be equipped with an additional expensive real-time monitoring device

[0007] The most representative TaqMan probe method (US Patent No. 5,210,015) and the self-quenching fluorescent probe method (US Patent No. 5,723,591) in real-time PCR have false positive results due to non-specific binding of double-labeled probes Therefore, it is actually difficult to realize the detection of 5-plex and must have skilled technology and skills as support

Because the existing PCR method needs to perform amplification and detection at the same time, the real-time PCR device has limited high-speed processing (High Throughput) capabilities.

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