Method and kit for detecting nucleic acid sequence of target to be detected by using melting curve of fluorescent probe

Abstract

The invention provides a method for detecting a to-be-detected target nucleic acid sequence by using a fluorescence probe melting curve and a kit thereof. The method comprises the following steps: designing a specific upstream primer, a specific downstream primer and a probe for each target nucleic acid sequence to be detected, so as to control the melting point Tm value of an amplification product of each target nucleic acid sequence to be detected and a probe binding fragment, the melting point Tm values of fragments formed by combining amplification products of different target nucleic acid sequences to be detected in the same fluorescence labeling channel with the probe are different, and the difference can be distinguished by a detection instrument. Multiple detection of multiple target nucleic acid sequences to be detected and / or detection of SNP in target nucleic acid sequences are / is realized through Tm difference and / or different fluorescent labels between PCR products of different target nucleic acid sequences to be detected and a binding fragment containing a fluorescent group on the right side of an RNA basic group.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for detecting target nucleic acid sequences to be detected by using a fluorescent probe melting curve and a kit thereof. Background technique [0002] With the demand for detection of multiple targets in a short period of time, such as the detection of multi-gene genetic loci, the detection of multi-infection targets in infectious syndromes, etc., how to achieve a single-tube reaction to detect or identify as many different target sequences or the same target as possible? The multi-site composition information of the sequence has become a key issue in the field of nucleic acid detection. [0003] Common multiple target detection technologies include gene chip technology, high-throughput sequencing technology, etc. Although they have the advantages of high throughput and high sensitivity, their high cost, professional analysis and expensive instruments still res...

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Problems solved by technology

[0005] At present, a variety of target sequence hybrid fluorescent probes have been used for melting curve analysis, including Taqman probes, molecular beacons and the melting curve technology (EMPA technology) invented by our company, etc., all of which can be labeled with different fluorescent groups In the melting curve analysis stage after the end of qPCR, it forms products with different Tm values ​​with fluorescence with different targets to realize the detection of multiple targets, but Taqman probes are prone to non-specific Tm peaks or even cannot form effective When detecting SNPs, the Tm value difference between the wild type and the mutant type is low, and it is not easy to distinguish; the shortcoming of the low sensitivity of molecular beacons also limits its clinical application

Although the EMPA technology invented by our company can achieve high-throughput in multiple detection, its discrimination between wild type and mutant type is also not high in SNP detection, and the existing technology cannot fully meet the clinical needs. Under such circumstances, we urgently need to develop a multiple melting curve detection technology with high sensitivity, high specificity and better SNP discrimination

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