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Quantitative PCR method adopting dye EvaGreen and dual HotStar polymerases

A hot-start enzyme and dye technology is applied in the field of quantitative PCR containing dye EvaGreen and dual hot-start enzymes to achieve the effect of inhibiting non-specific amplification, low price and high efficiency

Active Publication Date: 2015-12-23
上海科医联创医学检验所有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In summary, a high-efficiency, high-sensitivity, high-specificity and low-cost quantitative PCR method is needed, and there is no report about the quantitative PCR method of the present invention

Method used

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  • Quantitative PCR method adopting dye EvaGreen and dual HotStar polymerases
  • Quantitative PCR method adopting dye EvaGreen and dual HotStar polymerases
  • Quantitative PCR method adopting dye EvaGreen and dual HotStar polymerases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] In the real-time fluorescent quantitative PCR method, EvaGreen and the double hot start enzyme system have good repeatability, high sensitivity and precision.

[0056] (1) Construct a plasmid containing the β-globin gene and amplify the β-globin gene.

[0057] (2) Primer synthesis:

[0058] Forward primer: 5'-GAAGAGCCAAGGACAGGTAC-3'

[0059] Reverse primer: 5'-CAACTTCATCCACGTTCACC-3'

[0060] (3) Prepare the plasmid template as 3x10 6 , 3x10 5 , 3x10 4 , 3x10 3 , 3x10 2 , 3x10 1 , 3x10 0 The amount of template for each molecule, NTC is no substrate control, real-time fluorescence quantitative PCR reaction is carried out by using forward primer and reverse primer together with the PCR reaction master solution containing EvaGreen and dual hot start enzyme.

[0061] (4) Establishment of the PCR reaction system, the reaction system is 20 μL in total, and each reagent is added referring to the table below.

[0062] Table 1 PCR reaction system

[0063]

[0064] ...

Embodiment 2

[0072] In the real-time fluorescent quantitative PCR method, the dual hot start enzyme system has stronger sensitivity and higher amplification efficiency.

[0073] (1) Construct a plasmid containing the β-globin gene and amplify the β-globin gene.

[0074] (2) Primer synthesis is the same as in Example 1.

[0075] (3) Prepare the plasmid template as 3x10 4 , 3x10 3 , 3x10 2 , 3x10 1 , 3x10 0 The template amount of each molecule was amplified by dual hot-start enzyme and single hot-start enzyme respectively, and real-time fluorescent quantitative PCR reaction was carried out in a system with other components unchanged.

[0076] (4) PCR reaction system: dual hot-start enzymes, chemically modified DNA polymerase and antibody-modified DNA polymerase were used as hot-start enzymes, and other components were the same as in Example 1.

[0077] (5) qPCR reaction is the same as in Example 1.

[0078] (6) Experimental results

[0079] The result is as Figure 4 shown. Figure...

Embodiment 3

[0081] In the real-time fluorescent quantitative PCR method, EvaGreen has higher specificity and better repeatability.

[0082] (1) Prepare DNA to be tested; extract DNA from human blood according to conventional methods, and amplify uridine diphosphate glucuronosyltransferase gene (UGT1A1).

[0083] (2) Primer synthesis:

[0084] Forward primer: 5'-ACCTCTAGTTACATAACCTGA-3'

[0085] Reverse primer: 5'-AATAAACCCGACCTCACCAC-3'

[0086] (4) PCR reaction system: EvaGreen, SYBRGreenI and GelGreenI were used as fluorescent dyes, and other components were the same as in Example 1.

[0087] (5) The qPCR reaction procedure is the same as in Example 1.

[0088] (6) Experimental results

[0089] In this example, the difficult-to-amplify UGT1A1 gene tested by the inventors was selected as a template, and three double-stranded DNA-binding dyes, EvaGreen, SYBRGreenI and GelGreenI, were used to compare their specificity and repeatability. As shown in Figure 5. Figure 5A-1 and 5A-2 The ...

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Abstract

The invention relates to a quantitative PCR method adopting a dye EvaGreen and dual HotStar polymerases. The quantitative PCR method is characterized by comprising the following steps that 1, a PCR reaction system, including a sample to be tested, a forward primer and a reverse primer complementary with templates to be tested in the sample to be tested, the fluorescent dye EvaGreen and the dual HotStar polymerases including a HotStar Taq DNA polymerase and an Anti Taq DNA polymerase, is established; 2, PCR amplification is conducted on the templates to be tested in the PCR reaction system to form a double-stranded DNA-EvaGreen fluorescent dye compound; 3, a fluorescence value of the PCR reaction system is detected during or after the PCR amplification to determine the number of amplification products so as to determine the number of templates to be tested. The quantitative PCR method has the advantages of being high in accuracy, high in efficiency, high in flexibility, high in specificity and low in price.

Description

technical field [0001] The invention relates to a quantitative PCR method, in particular to a quantitative PCR method containing dye EvaGreen and dual hot start enzymes. Background technique [0002] Real-time fluorescence quantitative PCR (QuantitativeReal-timePCR) is the most advanced nucleic acid molecular diagnostic technology used in clinical practice in the world today. It is recognized and respected by the US FDA. The technology of real-time monitoring of the progress of the PCR reaction and the detection and analysis of the PCR reaction through the analysis software. The difference between qPCR technology and conventional PCR technology is that conventional PCR is cumbersome to operate and has poor specificity. After PCR, agarose gel electrophoresis is required for detection; while qPCR is detected during the reaction and does not require post-electrophoresis. Therefore, qPCR has the advantages of simple operation and short detection time. Short, not easy to be cont...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2561/101C12Q2545/114C12Q2531/113
Inventor 韩武梅吴国祥刘振柴勋张大挺梁乐
Owner 上海科医联创医学检验所有限公司
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