Reagent for enhancing real-time fluorescent PCR signal and method thereof

A technology of real-time fluorescence and enhanced fluorescence, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms. The effect of small values ​​and high signal values

Pending Publication Date: 2021-06-22
HANGZHOU KMB BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a real-time fluorescent PCR enhancer to overcome the high Ct value caused by the high GC content and the secondary structure of the hairpin loop caused by the high GC content, weak fluorescent signal and high non-specific signal, etc. question

Method used

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  • Reagent for enhancing real-time fluorescent PCR signal and method thereof
  • Reagent for enhancing real-time fluorescent PCR signal and method thereof
  • Reagent for enhancing real-time fluorescent PCR signal and method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1, a preparation method of the enhancer

[0022] The enhancer feedstock includes betaine and dimethyl sulfoxide, wherein the sugar base has a concentration of 5 m, a solvent is water, and the dimethyl sulfone concentration is 100%. The formulation method is mixed with 5M betaine with dimethyl sulfoxide.

Embodiment 2

[0023] Example 2 Application in a high GC content gene locus

[0024] 1, DNA sequence to be expanded

[0025] The GC content of this DNA region is 76%, and the bracket is to be measured.

[0026]

[0027] 2, primer sequence and probe sequence

[0028] The primers and probes described in Table 1 are designed in accordance with the DNA sequence to be amplified.

[0029] Table 1: Primers and probe sequences of real-time fluorescent PCR

[0030] Primers and probes sequence (5'-3 ') GC content (%) TM value (° C) Positive primer: gctgcagcggggcagcgg 82 68 Reverse primers: cgaggcgcacccgcagcc 82 69 Wild-type probe: Fam-gacgtgtgcgggccgc-mgb 81 76 Mutant probe: Vic-gacgTGCGCGCCG-MGB 86 75

[0031] 3, real-time fluorescent PCR reaction system and procedures

[0032] Real-time real-time fluorescent PCR reaction system is formulated according to Table 2.

[0033] Table 2

[0034] Component Volume in each reaction system TAQ enzyme (6U / μL)...

Embodiment 3

[0055] Example 3, Reaction System Stability Test

[0056]1, DNA sequence to be expanded

[0057] The GC content of this DNA region is 76%. In square brackets for the site.

[0058]

[0059] 2, primer sequence and probe sequence

[0060] The primers and probes of Table 7 were designed in response to the DNA sequence to be amplified.

[0061] Table 7: Real-time fluorescent PCR primers and probes sequences

[0062]

[0063] 3, real-time fluorescent PCR reaction system and procedures

[0064] In the PCR reaction system, a reinforcing agent including betaine and dimethyl sulfoxide is added, and the final concentration of the betaine is 0.6m, the final concentration of dimethyl sulfoxide is 1.5% (volume percent). Specifically, see Table 5.

[0065] table 5

[0066] Component Volume in each reaction system TAQ enzyme (6U / μL) 0.5μL DNTP (10mm) 0.4μL PCR buffer (2 ×) 10μL Positive primer (100 μm) 0.1μL Reverse primer (100 μm) 0.1μL Wild-type pro...

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PUM

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Abstract

The invention provides a reagent for enhancing fluorescent PCR signals and an application thereof. The reagent comprises an enhancer containing betaine and dimethyl sulfoxide. When a gene with relatively high GC content near a to-be-detected site is detected, the reagent and the method provided by the invention can enable genotyping of a gene site in a high-GC-content region to be more effective, specifically, a Ct value is smaller, a signal value is higher, and a non-specific signal is lower.

Description

Technical field [0001] The present invention relates to the field of biotechnology, and more particularly to reagents compositions and methods for enhancing fluorescent PCR signals. Background technique [0002] Real-time fluorescent PCR technology, by introducing specific probes and monitors fluorescent signals in real time to monitor the amplification of PCR products. Compared to sequencing, gene chip method, allele-specific PCR technology, high resolution melting curve method, real-time fluorescent PCR full process can be closed in single tube, and can automatically analyze the results, high sensitivity, accurate It is characterized by simple and fast operation. It is an advanced molecular detection technology. At present, gene polymorphism detection, microbial pathogens such as viruses is increasingly widely used with real-time fluorescent PCR method. [0003] There is a wide range of DNA sequences in the genome, which are particularly prone to the secondary structure interfe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686
CPCC12Q1/686
Inventor 丁佳女郑宜文周旭
Owner HANGZHOU KMB BIOTECH
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