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Universal fluorescent probe and detection method and application thereof

A fluorescent probe and detection method technology, applied in the field of general fluorescent probes and its detection, can solve the problems of easy pollution of the detection environment, easy misjudgment, and inability to realize real-time detection

Inactive Publication Date: 2017-05-10
GENOWISE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of this technology include: a) real-time detection cannot be realized; b) the analysis of the map results is difficult, and it is easy to misjudge; c) PCR products need post-processing, which is easy to pollute the detection environment
This molecular beacon technology needs to design specific probes for the target gene or mutation site, and different genes or mutation sites need to be redesigned and synthesized, which has high cost and time-consuming problems; in addition, it needs to be detected by the Tm peak of the melting curve , the detection sensitivity is limited

Method used

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  • Universal fluorescent probe and detection method and application thereof

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Experimental program
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Effect test

Embodiment 1

[0060] A preferred embodiment of the present invention provides a group of universal fluorescent probes, such as Figure 5 As shown, it includes positive-strand probe, negative-strand probe and specific downstream primer; wherein, positive-strand probe includes oligonucleotide positive-strand sequence (UAP) connected in sequence from 5' end to 3' end, ligation Sequence (TA Box) and specific upstream primer sequence (ASO); negative strand probe includes oligonucleotide negative strand sequence (UP) complementary to oligonucleotide positive strand sequence, oligonucleotide negative strand The 5' end and 3' end of the sequence are respectively connected with a fluorescent group (R) and a quencher group (Q). The fluorescent group can be selected from FAM, TET, Texas Red, VIC or Cy5, and the quencher group can be selected from BHQ1 Or BHQ2; the specific upstream primer sequence (ASO) and the specific downstream primer (LSO) are complementary to the target sequence of the target gen...

Embodiment 2

[0087] The present invention provides a method for genotyping the rs2294008 site by using a general-purpose fluorescent probe in a real-time fluorescent PCR instrument, and the specific steps are as follows:

[0088] S1. Synthesize positive-strand probes (allele-specific primer 1, allele-specific primer 2), and negative-strand probes complementary to the positive-strand probe (allele-specific primer 1, allele-specific primer 1, allele-specific primer 2). Sexual primers 2) and specific downstream primers (LSO);

[0089] Table 3 rs2294008 genotyping detection primer design table

[0090]

[0091]

[0092] In the table, the underlined part is the oligonucleotide positive strand sequence (UAP), the box marked is the connecting sequence (TA Box), and the unmarked part is the specific upstream primer sequence (ASO), corresponding to the detection site of rs2294008 wild type and mutant type.

[0093] S2. Selecting a human saliva sample and extracting the genomic DNA of the sa...

Embodiment 3

[0099] The present invention provides a method for genotyping the rs6010620 locus by using a general-purpose fluorescent probe in a real-time fluorescent PCR instrument, and the specific steps are as follows:

[0100] S1. Synthesize positive-strand probes (allele-specific primer 1, allele-specific primer 2), and negative-strand probes complementary to the positive-strand probe (allele-specific primer 1, allele-specific primer 1, allele-specific primer 2). Sexual primers 2) and specific downstream primers (LSO);

[0101] Table 4 Design table of primers for rs6010620 genotyping detection

[0102]

[0103]

[0104]In the table, the underlined part is the oligonucleotide positive strand sequence (UAP), the box marked is the connecting sequence (TA Box), and the unmarked part is the specific upstream primer sequence (ASO), corresponding to the detection of rs6010620 site wild type and mutant type.

[0105] S2. Selecting a human saliva sample and extracting the genomic DNA o...

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Abstract

The invention relates to the field of real-time fluorescent PCRs (Polymerase Chain Reactions), in particular a universal fluorescent probe and a detection method and an application thereof. The universal fluorescent probe comprises a positive chain probe, a negative chain probe and a specific downstream primer, wherein the positive chain probe comprises an oligonucleotide positive chain sequence, a connection sequence and a specific upstream primer sequence which are connected in sequence from end 5' to end 3'; the negative chain probe comprises an oligonucleotide negative chain sequence which is complementary with the oligonucleotide positive chain sequence, the end 5' and the end 3' of the oligonucleotide negative chain sequence are connected with a fluorescent group and a quenching group respectively; the specific upstream primer sequence and the specific downstream primer are complementary with a target sequence of a target gene. According to the detection method, fluorescent PCR amplification is performed by the universal fluorescent probe; the universal fluorescent probe can be applied to genetic typing and mutation gene detection; by adopting the universal fluorescent probe and the detection method and the application thereof, real-time detection is realized, PCR subsequent treatment is not required, the designing and synthesizing time is saved, cost is lowered, and high result stability and easiness in analysis are realized.

Description

technical field [0001] The invention relates to the field of real-time fluorescent PCR, in particular to a universal fluorescent probe and its detection method and application. Background technique [0002] In the prior art, the application of the fluorescein labeling method in PCR is mainly reflected in two aspects of fluorescent primers or fluorescent probes. [0003] Among them, the method of fluorescent primers mainly includes direct labeling primers or labeling adapter primers, and PCR products use genetic analyzers, such as ABI genetic analyzers 310, 3130, 3130xl, 3730, etc., which are usually used for detecting microsatellites and genotyping. For example, the Chinese patent announcement number is the invention patent of CN104178566, which discloses a kind of multiple fluorescent PCR universal connector and detection method and application that can be used for microsatellite detection. Its primer design diagram and detection flow chart are respectively as follows figu...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6858C12Q2531/113C12Q2563/107
Inventor 周立新叶绍云白永飞李丹杨小娟蒋峻峰
Owner GENOWISE
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