Method for detecting by using labeled probe and analyzing fusion curve

Abstract

The invention relates to a method for detecting by using labeled probe and analyzing a fusion curve and a kit comprising the probe, and belongs to the field of biotechnology. In the method, an amplification system comprises thermostable DNA polymerase without 5' nuclease activity, a labeled oligonucleotide probe, and other common components in the amplification system, wherein the labeled probe comprises a report label and a quenching label; and because of the DNA polymerase, the probe cannot be hydrolyzed; therefore, the probe can be designed to have a long length so as to cover a large area comprising a plurality of mutated nucleotide target sequences and overcome the defect of short labeled probe in the prior art. The method and the kit can simply and conveniently detect the target sequences and analyze the fusion curve, can save cost and detect the target sequences comprising various mutations.

Description

technical field [0001] The invention relates to the technical field of nucleic acid detection and melting curve analysis using labeled probes, in particular to a method for analyzing target nucleic acid sequences containing at least one mutated nucleotide using double-labeled oligonucleotide probes. Background technique [0002] Real-time PCR can be used to detect and quantify target nucleic acid sequences. Nowadays, many probe-based methods are used in real-time PCR, such as TaqMan probes (U.S.Pat.Nos.5,210,015 and 5,487,972), molecular beacon probes (molecular beacons, U.S.Pat.Nos.5,925,517 and 6,103,476), Self-probing amplicons (self-probing amplicons), also known as scorpions (U.S.Pat.No.6,326,145), Amplifluor (Chen et al., Appl.Environ.Microbiol.64:4210-6, 1998), Amplifluor (U.S.Pat .No.6,117,635), displacement hybridization probe (Li et al., Nucleic Acids Res.30:E5, 2002), DzyNA-PCR (Todd et al., Clin.Chem.46:625-30, 2000), fluorescence Restriction enzyme detection (...

AI Technical Summary

Problems solved by technology

However, the detection method of the labeled TaqMan probe in this report also has its limitations

First, it uses Taq polymerase with 5' nuclease activity. In order to prevent the hydrolysis of the probe in the conventional TaqManreal-time PCR extension step, the melting temperature (Tm value) of the selected probe should be higher than the Tm value of the PCR primer. 10°C lower, due to the lower Tm value of the probe, the hybridization efficiency of the probe is also lower, and the final fluorescence detection signal is also weaker, and the lower Tm value of the probe means that the probe is shorter. This makes it impossible for the probe to cover a long distance to the target sequence, which is very unfavorable for detecting a series of mutations in the core region of a gene, such as the core region of the RpoB gene in TB drug resistance; secondly, based on TaqMan probes Melting curve analysis is generally not suitable for long probes, because the reporter label and quencher label are separated by long-distance nucleotides, so that the reporter label and quencher label cannot interact

For a long TaqMan probe, it is difficult to detect the difference in fluorescence intensity between the hybridized state and the single-stranded state of the probe

Among the existing technical methods, the TaqMan real-time PCR method can detect the fluorescent signal in real time at each cycle, but usually cannot perform melting curve analysis

The improved TaqMan-based method reported by Housni et al. is capable of melting curve analysis when the probe encounters suitable conditions, but cannot detect the fluorescence signal in real time at each cycle.

Molecular signal probe-based methods can detect fluorescent signals in real time at each cycle, but cannot perform melting curve analysis of mixtures of probes hybridized to target sequences

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