Incision enzyme mediated real-time multiple cross nucleic acid displacement amplification technology and application

A technology of restriction endonucleases and amplification primers, which can be used in the determination/testing of microorganisms, biochemical equipment and methods, etc., and can solve problems such as expensive, time-consuming and labor-consuming, and poor diagnostic sensitivity

Active Publication Date: 2016-07-13
ICDC CHINA CDC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0008] At present, the detection of Shigella and Salmonella 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. , the interpretation of biochemical results depends on human subjective judgment, resulting in poor repeatability of results and easy misjudgment
With the rapid development of nucleic acid diagnostic technology, some PCR-based diagnostic technologies (such as ordinary PCR te

Method used

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  • Incision enzyme mediated real-time multiple cross nucleic acid displacement amplification technology and application
  • Incision enzyme mediated real-time multiple cross nucleic acid displacement amplification technology and application
  • Incision enzyme mediated real-time multiple cross nucleic acid displacement amplification technology and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] Embodiment 1. Standard ET-MCDA detects

[0066] 1.1 Establish a standard ET-MCDA reaction system: the concentration of cross primers E-CP1 and CP1 is 30pmol, the concentration of cross primer CP2 is 60pmol, the concentration of replacement primers F1 and F2 is 10pmol, the concentration of amplification primers R1, R2, D1 and D2 The concentration is 30pmol, the concentration of amplification primers C1 and C2 is 20pmol, 12.5μL 2×Mix buffer reaction solution, 10U strand displacement DNA polymerase (Bst), 15U restriction endonuclease (Nb.BsrDI), 1μL template , add deionized water to 25 μl. The entire reaction was kept at 63°C for 1 hour, and the reaction was terminated at 80°C for 5 minutes.

[0067] 1.2 Prove the feasibility of the designed MCDA amplification reaction

[0068] Visible color change method: Under standard ET-MCDA reaction conditions, ET-MCDA generates a large amount of pyrophosphate ions when amplifying DNA, which can capture manganese ions bound to calce...

Embodiment 2

[0075] Embodiment 2. Multiple ET-MCDA detection

[0076] In order to obtain stable multiple fluorescence detection patterns, the standard ET-MCDA system was optimized to adapt to multiple detection, and a multiple detection system was established. Under the multiplex detection system, we added two sets of primers and corresponding templates to the multiplex reaction mixture at the same time, detected by a fluorescence detector, and obtained a stable multiplex fluorescence detection map, as shown in Figure 7 (signals of group A and group B are generated simultaneously, From the same reaction tube. Group A signals come from the Cy5 signal channel, representing the detection of Salmonella, signals 1 to 12 represent the amount of detection samples are 25ng, 2.5ng, 250pg, 25pg, 2.5pg, 250fg, 25fg, 12.5fg, 6.25 fg, 3.125fg, 1.56fg to 0.78fg / reaction tube, signal 13 represents the negative control; group B signals come from the HEX channel, representing the detection of Shigella, sig...

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Abstract

The invention discloses an incision enzyme mediated real-time multiple cross nucleic acid displacement amplification technology and an application. The invention provides a pair of cross primers which contain restriction incision enzyme specific recognition sequences at an end 5', and are marked with fluorescent groups and quenching groups, a pair of displacement primers and 3-6 amplification primer constant-temperature amplification target gene segments; and the incision enzyme mediated real-time multiple cross nucleic acid displacement amplification technology can carry out visible detection, electrophoresis detection, real-time turbidity detection and real-time fluorescence detection. The method is convenient, quick, sensitive, specific and suitable for detecting various nucleotide fragments.

Description

technical field [0001] The invention discloses a nucleic acid amplification technology, which belongs to the field of molecular biology. Background technique [0002] In the field of modern medicine and biology, 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 using PCR technology for nucleic acid amplification, it relies on complex and expensive thermal cycle equipment, and the detection of amplification products is relatively complicated, requiring a complex process and equipment. Therefore, the applic...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6844C12Q2531/119C12Q2563/107C12Q2561/113C12Q2521/101C12Q2521/301
Inventor 叶长芸王毅王艳
Owner ICDC CHINA CDC
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