Composition for detection and detection method

A detection method and composition technology, applied in the field of molecular biology, can solve problems such as high cost, time-consuming and labor-intensive, and inability to meet the quantitative requirements of the transgenic labeling system

Inactive Publication Date: 2019-03-01
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The traditional MLPA technology relies on capillary electrophoresis as a means of product detection, and draws conclusions by analyzing the changes in the fragment peak position and peak area through capillary electrophoresis results. The operation is cumbersome, time-consuming and laborious.
Some studies have improved the traditional method and adopted the method of labeling fluorescence to make it easier to identify electrophoretic peaks, but this method is costly
And none of them can accurately quantify the target sequence, and cannot meet the quantitative needs of various countries for the GMO labeling system

Method used

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  • Composition for detection and detection method
  • Composition for detection and detection method
  • Composition for detection and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Example 1. Establishment of a novel fluorescent multiplex ligation-dependent probe amplification technology system F-MLPA

[0076] (1) Design of ligation probe sequence

[0077] According to the whole or partial sequence of the target nucleotide sequence to be detected or to be amplified (hereinafter referred to as sequence A), obtain a nucleotide sequence complementary to the target nucleotide sequence to be detected or to be amplified (hereinafter referred to as sequence C) ), the complementary binding nucleotide sequence is divided into two parts, that is, two nucleotide sequences (hereinafter referred to as sequence C1 and sequence C2, it should be pointed out that sequence C1 and sequence C2 have no sequence, but simply Used to represent the different parts in the two parts, or the different nucleotide sequences in the two nucleotide sequences), one part (sequence C1) is used to design the upstream connection probe, and the other part (sequence C2) Used to design ...

Embodiment 2

[0122] Embodiment 2, condition optimization experiment

[0123] (1) Optimization of the DNA denaturation process during the ligation probe reaction

[0124] The single-strand preparation process, that is, the DNA denaturation process in the kit manual of the Dutch MRC-Holland company is 95°C for 5 minutes, and then cooled to 25°C.

[0125] In Example 1, the DNA denaturation process was changed to 98°C for 5 minutes, and then quickly placed in ice water for 20 minutes.

[0126] Using the transgenic crop MON810 as the template and MON810 as the target gene, the DNA denaturation process described above was compared and optimized. 2% agarose gel electrophoresis detection results.

[0127] DNA denaturation process optimization results such as figure 2 shown. From figure 2 It can be seen from the figure that if the DNA denaturation process is changed to 98°C for 5 minutes, and then quickly placed in ice water, the amplified product of the ligation product is clearer.

[0128...

Embodiment 3

[0134] Embodiment 3, single weight and multiple feasibility verification experiments

[0135] (1) Single feasibility verification experiment

[0136] The feasibility of multiple pairs of connection-dependent probes shown in Table 2 in Example 1 was verified respectively. The template and connection probe sequences in each reaction system of the feasibility verification experiment were adjusted according to Example 1, and the others were consistent with Example 1. Finally, the experimental results were detected by 2% gel electrophoresis.

[0137] The results of the single-fold feasibility verification experiment are as follows: Figure 5 shown. From Figure 5 It can be seen that the four groups of probes (Mon810-2F and Mon810-2R, ZSS-2F and ZSS-2R, GTS-2F and GTS-2R, LEC-2F and LEC-2R) are all effectively connected and the connection products can be used in general PCR amplification was carried out under the guidance of primers, and the fragment sizes of the amplified produ...

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Abstract

The invention provides a composition for detection and a detection method. A filling sequence in traditional MLPA (Multiplex Ligation-dependent Probe Amplification) is replaced with one section of a sequence consistent to a fluorescent probe sequence. An ingenious design is adopted and an F-MLPA method established by the invention is used for changing an existing MLPA method into a quantitative and visual detection technology; a designed fluorescent probe only can be combined with a PCR (Polymerase Chain Reaction) product of a probe ligation product and a false positive phenomenon is avoided.In one specific embodiment, the probe sequence and a Taqman fluorescent probe which are designed by the invention, and the F-MLPA method established by the invention are adopted to finally and successfully realize specific detection of transgenic maize MON810 and transgenic soybean GTS 40-3-2; the detection sensitivity reaches 1nM. Moreover, the F-MLPA method and system, which are established by the invention, are of a multi-PCR reaction system for carrying out quantitative detection on exogenous specific genes and reference genes at the same time; accurate and quantitative detection of transgenic components can be realized. The invention provides a novel platform and concept for researching and developing the detection product or method.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a detection composition and a detection method. Background technique [0002] In 2016, the International Service for the Acquisition of Agri-Biotech Applications (ISAAA) published the report "2016 Global Biotechnology / Transgenic Crops Commercialization Development Trends" which pointed out that from 1996 to 2016, transgenic crops The planted area of ​​China has grown rapidly from 1.7 million hectares to 185.4 million hectares. In 2016, the global biotech crop planting area increased by 5.7 million hectares relative to the 179.7 million hectares in 2015. With the rapid development of genetically modified technology, safety issues such as toxicity, drug resistance, and allergies of genetically modified foods have attracted more and more public attention, and some debates about genetically modified foods have even affected public attitudes towards genetically ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q2561/113C12Q2563/107C12Q2545/114C12Q2561/101C12Q2537/143C12Q2531/113
Inventor 许文涛罗云波牛晨启黄昆仑徐瑗聪杜再慧贺晓云
Owner CHINA AGRI UNIV
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