ET-PCR (endonuclease restriction-mediated real-time polymerase chain reaction) nucleic acid testing technology

A technology of restriction endonuclease and amplification system, which is applied in the determination/inspection of microorganisms, methods based on microorganisms, biochemical equipment and methods, etc., and can solve the problems of poor repeatability of results, easy misjudgment, time-consuming and labor-intensive, etc. question

Inactive Publication Date: 2016-10-26
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the diagnosis of Streptococcus pneumoniae, Staphylococcus aureus and Enterococcus faecalis mainly relies on traditional enrichment culture and biochemical identification. This method takes about 5 to 7 days, including enrichment, selective culture and subsequent biochemical identification. Its disadvantage is time-consuming and labor-intensive, and the interpretation of biochemical

Method used

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  • ET-PCR (endonuclease restriction-mediated real-time polymerase chain reaction) nucleic acid testing technology
  • ET-PCR (endonuclease restriction-mediated real-time polymerase chain reaction) nucleic acid testing technology
  • ET-PCR (endonuclease restriction-mediated real-time polymerase chain reaction) nucleic acid testing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Embodiment 1 standard ET-PCR reaction

[0060] 1. Standard ET-PCR reaction system:

[0061]

[0062] 2. Standard ET-PCR reaction amplification parameters

[0063] Amplify and measure the results on a real-time fluorescent quantitative PCR instrument:

[0064]

[0065] The Real-time reaction was carried out in a fluorescence detector (Rotor-Gene Q Real Time System, Qiagen). After the amplification was completed, the background fluorescence signal was subtracted and the data was analyzed at the same threshold to determine the Ct (cyclethreshold) value of the reaction.

[0066] 3. Feasibility of ET-PCR amplification reaction

[0067] (1) Fluorescence detection method: Under standard ET-PCR reaction conditions, ET-PCR generates a large number of fluorescent signals when amplifying DNA, which can be detected by a real-time fluorescence detector and generate stable fluorescent signals. The intensity correlates positively with the product of the amplicon, see Figure...

Embodiment 2

[0078] Embodiment 2: multiplex ET-PCR reaction

[0079] 1. Multiplex ET-PCR reaction system:

[0080]

[0081] 2. Multiplex ET-PCR amplification parameters

[0082] Amplify and measure the results on a real-time fluorescent quantitative PCR instrument:

[0083]

[0084]

[0085] The Real-time reaction was carried out in a fluorescence detector (Rotor-Gene Q Real Time System, Qiagen). After the amplification was completed, the background fluorescence signal was subtracted and the data was analyzed at the same threshold to determine the Ct (cyclethreshold) value of the reaction.

[0086] 3. The multiple detection ability of ET-PCR

[0087] In order to obtain stable multiple fluorescence detection patterns, the standard ET-PCR system was optimized to adapt to multiple detection, and a multiple detection system was established. Under the multiplex detection system, we added three sets of primers and corresponding templates to the multiplex reaction mixture at the same ...

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Abstract

The invention discloses a PCR (polymerase chain reaction) amplification method. In the method, a restriction endonuclease sequence is added to a 5'end of an upstream primer or a downstream primer for amplification, a fluorophore is marked at the 5'end of the primer, a quenching group capable of specifically quenching the fluorophore at the 5'end is marked in the middle of the primer, the restriction endonuclease is added to a PCR amplification system and subjected to a PCR. The method has the advantages that the amplification speed is high, the reaction is sensitive, the specificity is high, and real-time, rapid and multiplex detection and analysis of nucleic acid can be realized.

Description

technical field [0001] The invention relates to a nucleic acid amplification method, which belongs to the field of microorganisms and molecular biology. Background technique [0002] In the field of modern biology and medicine, nucleic acid amplification is an indispensable technology, widely used in clinical testing, basic research, archaeological research, epidemiological research, transgenic research and other fields. Among the nucleic acid amplification technologies that have been developed and designed, PCR amplification technology is the first established in vitro nucleic acid amplification technology, which has epoch-making significance. This technology has been widely used in medical, biological and chemical related fields. [0003] When PCR technology is used for nucleic acid amplification, three thermal cycle steps need to be repeated: nucleic acid melting (95°C), primer annealing (45-65°C), and extension amplification (72°C). After the PCR amplification is comple...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/14C12Q1/10C12R1/46C12R1/445
CPCC12Q1/686C12Q1/689C12Q2521/301C12Q2563/107
Inventor 叶长芸王毅王艳
Owner ICDC CHINA CDC
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