FRET-based fluorescent nucleic acid detection method and kit

A technology for detecting kits and fluorescent nucleic acids, applied in the field of real-time fluorescent quantitative PCR, to achieve the effects of wide application, increased design success rate, and elimination of primer-dimers

Pending Publication Date: 2018-10-12
CARRIER GENE TECH SUZHOU CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0012] But above-mentioned real-time fluorescence quantitative PCR technique still has respective shortcoming, therefore, the present invention provides a kind of resonance energy transfer between the primer pair based on fluorescent donor group (D) and acceptor group (A) double labeling (FRET) mechanism fluorescent nucleic acid detection method and kit, named Primobe TM , for nucleic acid real-time fluorescence quantification (real-timequantification), belongs to the technical field of real-time fluorescence quantitative PCR

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  • FRET-based fluorescent nucleic acid detection method and kit
  • FRET-based fluorescent nucleic acid detection method and kit

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Embodiment 1

[0035] A FRET-based fluorescent nucleic acid detection method, the kit contains: one or more sets of Primobe that are respectively labeled with a fluorescent donor group and a fluorescent acceptor group and can be used for real-time fluorescence quantification of nucleic acids TM ;PCR reaction mother solution (Master Mix) containing thermostable DNA polymerase, dNTP, PCR buffer; Deionized water; Standard substance of known concentration; Negative quality control sample; Positive quality control sample;

[0036] The Primobe TM One or more sets of oligonucleotide pairs labeled with fluorescent donor groups and fluorescent acceptor groups, which can be used for real-time fluorescence quantification of nucleic acids; each group is used for real-time fluorescence quantitative nucleic acid Primobe TM Consists of 2 primers; Primobe TM Any position of one of the primers is labeled with a fluorescent acceptor group (Accepter, A); Primobe TM A fluorescent donor group (Donor, D) can be...

Embodiment 2

[0048] 1. Taking the detection of human SRY gene as an example, design and synthesize a set of fluorescently labeled Primobe TM Primer, its sequence is as follows:

[0049] Upstream primer SRYf: 5'-CGATCAGAGGCGCAAGATGGCT(FAM)C-3'.

[0050] Downstream primer SRYr: 5'-TCTCTGAGTTTCGCATTCTGGGATTCTCT(LC-Red640)A-3'.

[0051] 2. Template and Primobe TM dilution:

[0052] 1) Thaw the genomic DNA of the unknown sample to be tested on ice, shake and mix well, and dilute to a final concentration of 5-50 ng / μL.

[0053] 2) SRYf and SRYr were respectively diluted with TE buffer to a final concentration of 2.5 μM.

[0054] 3. Preparation of PCR reaction system

[0055] 1) Use the 96-well optical reaction plate produced by ThermoFisher to perform real-time fluorescent quantitative PCR experiments. In the reaction wells planned for unknown sample detection, add the following components in sequence.

[0056]

[0057] 2) Unknown samples were tested 3 times to test the repeatability o...

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Abstract

The invention discloses an FRET-based fluorescent nucleic acid detection method and kit. The FRET-based fluorescent nucleic acid detection method comprises the following steps of 1) mixing template DNA, PrimobeTM, PCR reaction mother liquid, and using deionized water to supplement the total volume of a reaction as needed; 2) for real-time gene quantification testing, setting repeat reaction, standard gradient, positive control and negative control of unknown samples; 3) starting instruments and software, and setting PCR reaction parameters and fluorescence signal collection points; 4) performing PCR amplification and collecting signals during the reaction; 5) analyzing data. The target fluorescent nucleic acid detection method has the following advantages of simple scheme, strong specificity, quantitative precision and wide application, PrimobeTM is a versatile and basic quantitative PCR technology scheme design, and the method can be used for real-time fluorescence quantification, andcan also be used for identification of other types of target genes.

Description

technical field [0001] The invention relates to the technical field of real-time fluorescent quantitative PCR, in particular to a FRET-based fluorescent nucleic acid detection method and kit. Background technique [0002] In 1992, Higuchi proposed the idea of ​​real-time quantitative PCR. In 1996, Applied Biosystems of the United States realized the commercialization of real-time quantitative PCR technology. Real-time fluorescence quantitative PCR technology has great advantages: it realizes the leap from qualitative to quantitative PCR; the automatic real-time fluorescent quantitative PCR instrument is easy to operate, which makes genetic testing easy to standardize; closed-tube detection does not require such methods as agarose gel electrophoresis Wait for post-PCR processing to avoid cross-contamination. Real-time fluorescent quantitative PCR technology is widely used, including quantitative monitoring of pathogens and drug efficacy evaluation, gene single nucleotide va...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6876C12Q1/6818
CPCC12Q1/6818C12Q1/6851C12Q1/6876C12Q2531/113C12Q2561/113C12Q2563/107
Inventor 陈云弟李仁强陈苗苗
Owner CARRIER GENE TECH SUZHOU CO LTD
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