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Primers for detection of transgenic alfalfa j101 strain-specific loop-mediated isothermal amplification, detection method and application

A transgenic alfalfa, ring-mediated isothermal technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc. Highly specific, sensitive, and time-consuming effects

Active Publication Date: 2017-11-03
黄埔出入境检验检疫局综合技术服务中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The technical problem to be solved in the present invention is to provide a group of transgenic alfalfa J101 strain-specific LAMP detection primers for the screening detection technology used in the existing transgenic alfalfa that cannot accurately detect specific transgenic strains. High specificity and sensitivity, based on the primers and their combination, the present invention can successfully establish a visual detection method for transgenic alfalfa J101 strain-specific LAMP

Method used

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  • Primers for detection of transgenic alfalfa j101 strain-specific loop-mediated isothermal amplification, detection method and application
  • Primers for detection of transgenic alfalfa j101 strain-specific loop-mediated isothermal amplification, detection method and application
  • Primers for detection of transgenic alfalfa j101 strain-specific loop-mediated isothermal amplification, detection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Design of primers and screening and LAMP detection system components

[0046] The present invention combines a large number of experimental studies through creative analysis, and is based on the sequence of the contiguous region between the 3' end foreign insert fragment E9 3 of the transgenic alfalfa J101 line and the alfalfa genomic DNA, see the attached figure 1 Shown in A. Combined with the analysis of sequence information, LAMPdesigner software was used to design and obtain a set of specific LAMP primers, see the attached figure 2 ; To establish a specific and sensitive detection method for transgenic alfalfa J101 strain-specific LAMP.

[0047] The present invention attempts to design LAMP primers by using the 5' end Pe-FMV (promoter) end of the J101 strain genome and the adjacent region sequence of the alfalfa genome DNA, but no suitable LAMP primers can be obtained through bioinformatics analysis.

[0048] Another set of primers F3-2, B3-2, FIP(F1c+...

Embodiment 2

[0065] Example 2: Selection experiment of reaction temperature of J101 strain-specific LAMP detection system

[0066] This embodiment is based on the primers of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, comprising the following steps:

[0067] S1. Extract DNA as a template for the reaction; weigh about 100 mg of the sample to be tested and grind it into dry powder, and use the Tiangen Plant Genomic DNA Extraction Kit to extract genomic DNA according to its operating instructions. The concentration of the extracted genomic DNA was measured with a micro-spectrophotometer nanodrop2000c. The extracted DNA solution was stored at -20°C for future use.

[0068] S2. Use the primers SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 to perform visual detection of LAMP, and judge according to the detection results ;

[0069] Among them, the LAMP detection reaction system described in S2 is 25 μL: 10× Thermopol buffer...

Embodiment 3

[0072] Example 3: J101 strain-specific LAMP detection system Mg 2+ Concentration options:

[0073] This embodiment is based on the primers of SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6, comprising the following steps:

[0074] S1. Extract DNA as a template for the reaction; weigh about 100 mg of the sample to be tested and grind it into dry powder, and use the Tiangen Plant Genomic DNA Extraction Kit to extract genomic DNA according to its operating instructions. The concentration of the extracted genomic DNA was measured with a micro-spectrophotometer nanodrop2000c. The extracted DNA solution was stored at -20°C for future use.

[0075] S2. Use the primers SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.6 to perform visual detection of LAMP, and judge according to the detection results ;

[0076] Among them, the LAMP detection reaction system described in S2 is 25 μL: 10× Thermopol buffer (1×), F3 and B3 e...

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Abstract

The invention discloses primers and method for specific loop-mediated isothermal amplification (LAMP) detection of a genetically modified alfalfa strain J101 and an application of the method. The sequences of the primers are respectively shown in SEQ ID NO.1-SEQ ID NO.6. Aiming at the genetically modified alfalfa strain J101 from the Monsanto company to which the ministry of agriculture of China has not issued a security certificate of genetically modified organisms in agriculture, a group of specific primers are provided for the first time and a specific LAMP rapid detection system of the alfalfa strain J101 is successfully established by promoting LAMP with BstDNA polymerase and optimizing an LAMP detection system. The detection system is simple to operate, has strong specificity and high sensitivity, is accurate in detection results, has a minimum detection copy number of 10 and can be used for carrying out detection analysis and identification on the components of genetically modified alfalfa J101 rapidly and accurately.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a transgenic alfalfa J101 strain-specific loop-mediated isothermal amplification (LAMP) detection primer, detection method and application. Background technique [0002] The genetically modified alfalfa J101 strain is a herbicide-resistant genetically modified strain developed by Monsanto. In 2005, the United States and Canada approved its environmental release and application as feed. At present, it has not obtained the safety certificate of genetically modified organisms in my country, and its import has not yet been approved. [0003] Genetically modified alfalfa enters the food chain of animal breeding through feed, causing milk, meat and other products to become genetically modified food, which may have an impact on human health. According to the "Agricultural GMO Safety Management Regulations", "Agricultural GMO Import Safety Evaluation Management Measures", "Ag...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 刘二龙吕英姿卢丽蒋湘樊武疆姚柏辉王定国苏彩珠唐婕林惠娇郑高彬林学勤符其娇秦焯敏
Owner 黄埔出入境检验检疫局综合技术服务中心
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