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Universal probe technology based fluorescent quantitation PCR (Polymerase Chain Reaction) method

A general-purpose probe, fluorescence quantitative technology, applied in the field of optimization of fluorescent probe technology, can solve the problems of high cost of Taqman probe synthesis and increase of experimental cost, etc.

Active Publication Date: 2018-06-29
李保伟
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cost of Taqman probe synthesis is high. When detecting multiple sites or genes, multiple Taqman probes need to be synthesized, which will increase a lot of experimental costs, which is also the main reason for limiting the clinical promotion of fluorescent quantitative PCR technology.

Method used

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  • Universal probe technology based fluorescent quantitation PCR (Polymerase Chain Reaction) method
  • Universal probe technology based fluorescent quantitation PCR (Polymerase Chain Reaction) method
  • Universal probe technology based fluorescent quantitation PCR (Polymerase Chain Reaction) method

Examples

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Embodiment 1

[0019] The polymorphic site rs671[A / G] of ALDH2 is detected by the fluorescent quantitative PCR method of the terminal universal probe. The fluorescent quantitative PCR reaction process based on the terminal universal probe used in this embodiment is as follows figure 1 As shown, the reaction is divided into two stages.

[0020] The first stage is ordinary PCR amplification for polymorphic sites. Primers are designed according to the template sequence 1. The sequences of the upstream combined specific primer 2 are divided into two types: wild type and mutant type. See Table 1. SEQ ID NO.1 and 2 , see the sequence of downstream specific primer 3 in Table 1. SEQ ID NO.3. 20μL reaction system, including TaKaRa Taq HS (5 U / μl) 0.2μL, 10×PCR Buffer 2μL, dNTP Mixture (each 2.5mM) 1μL, three kinds in the system The primers are mixed at a molar ratio of 1:1:2, and the final concentration of each primer is between 200 and 500nM. Three human genomes with known genotypes after generation...

Embodiment 2

[0024] Utilize the fluorescent quantitative PCR method of middle universal probe to detect the content of Escherichia coli nucleic acid, the fluorescent quantitative PCR reaction process based on middle universal probe used in this embodiment is as follows image 3shown. Primers were designed according to the specific sequence 1 of the Escherichia coli genome, the sequence of the upstream specific primer 12 is shown in Table 1 SEQ ID NO.8, the sequence of the downstream specific primer 3 is shown in Table 1 SEQ ID NO.9, and the sequence of the intermediate combination specific primer 14 is shown in Table 1 SEQ ID NO.10, the sequence of the middle universal probe 13 is shown in Table 1. SEQ ID NO.11. In the 20 μL reaction system, the four primers are mixed at a molar ratio of 2:2:1:1, and the final concentration of each primer is between 100 and 400 nM , the final template concentrations were 1ng / 20μL, 0.1ng / 20μL, 0.01ng / 20μL, 0.001ng / 20μL, and a tube of negative control was ad...

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Abstract

The invention relates to the field of fluorescent quantitation PCR (Polymerase Chain Reaction), and provides a universal probe technology based fluorescent quantitation PCR method. The method has theadvantage of high Taqman probe specificity, low development cost and high popularization value. The method is characterized in that two universal probe technologies are the core parts; a probe in an end universal probe technology is a double-mark probe which complements the special sequence of an upstream combination specific primer; an intermediate specific primer has the effect similar to Taqmanprobe specificity and is capable of inhibiting the hydrolyzing and signal release of the probe; the probe in the intermediate universal probe technology is a 3' end single-mark fluorescent probe andcomplements the special sequence of an intermediate combination specific primer; the intermediate combination specific primer has the effect similar to Taqman probe specificity; a quenching group is marked at the 5' end of the primer; when the primer is hydrolyzed in PCR reaction, the quenching group is free, therefore, the signal of the probe can be released.

Description

technical field [0001] The present invention relates to the field of fluorescent quantitative PCR, in particular to the optimization of fluorescent probe technology. By using one or a few general probes combined with intermediate specific primers, the specificity of fluorescent quantitative PCR reaction can be achieved and the experimental cost can be reduced. Background technique [0002] Fluorescent quantitative PCR is a new nucleic acid quantitative experimental technology introduced by Applied Biosystems in 1996. Its principle is to label and track PCR products through fluorescent dyes or fluorescently labeled specific probes, and monitor the reaction process in real time. , through the real-time analysis of the product quantity, it is finally possible to make an accurate judgment on information such as the initial concentration of the template, the type of nucleic acid, and the type of gene mutation. At present, fluorescent quantitative PCR is widely used in the fields ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12Q1/06
CPCC12Q1/6851C12Q2537/143C12Q2563/107C12Q2531/113Y02A50/30
Inventor 李保伟
Owner 李保伟
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