Primer and probe for real-time fluorescent PCR detection of transgenic alfalfa J163 strains and detection method and applications thereof

A technology of transgenic alfalfa and J163, which is applied in biochemical equipment and methods, measurement/inspection of microorganisms, DNA/RNA fragments, etc., can solve the problems of inaccurate detection of transgenic strains, etc., and achieve stable detection and analysis, simple and stable operation sex high effect

Active Publication Date: 2015-06-03
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

[0008] The technical problem to be solved in the present invention is to provide a kind of primer and probe for transgenic alfalfa J163 strain-specific real-time fluorescent PCR detection aiming at the deficiency that the screening detection technology adopted by the existing transgenic alfalfa cannot accurately detect specific transgenic strains , which has high specificity, sensitivity and stability, based on the primers and probes, a specific real-time fluorescent PCR detection method for transgenic alfalfa J163 strains can be successfully established

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  • Primer and probe for real-time fluorescent PCR detection of transgenic alfalfa J163 strains and detection method and applications thereof
  • Primer and probe for real-time fluorescent PCR detection of transgenic alfalfa J163 strains and detection method and applications thereof
  • Primer and probe for real-time fluorescent PCR detection of transgenic alfalfa J163 strains and detection method and applications thereof

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Experimental program
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Effect test

Embodiment 1

[0050] Example 1: Establishment of J163 strain-specific real-time fluorescent PCR detection system

[0051] Design primers and probes:

[0052] The present invention combines a large number of experimental studies through creative analysis, based on the sequence of the contiguous region between the 5' exogenous insert fragment P-eFMV of the transgenic alfalfa J163 line and the alfalfa genomic DNA, combined with the analysis of sequence information, using press 3.0 software ( Applied Biosystems, USA) designed primers and probes. The designed primers and probes were compared by Blast in the NCBI website database to determine the theoretical specificity of the primers and probes. And targeted to determine the process conditions, the establishment of specificity, high sensitivity, good stability of transgenic alfalfa J163 strain-specific TaqMan real-time fluorescent PCR detection method.

[0053] The sequences of the primers J163-1 and J163-2 for the specific real-time fluoresce...

Embodiment 2

[0064] Embodiment 2: the specificity test of the inventive method

[0065] The present invention uses a variety of crops to carry out specificity experiments. The following 15 kinds of crops include J163, transgenic corn MIR162, transgenic corn Btll, transgenic corn MON98140, transgenic corn NK603, transgenic rice (cry IA (b / c)), transgenic soybean GTS40-3.2, non-GMO alfalfa, alfalfa non-GMO rapeseed, soybean, coconut meal, pea, cassava and wheat. (both stored after conventional random sampling, and therefore do not limit the scope of the present invention) genomic DNA as a template, the transgenic alfalfa J163 strain-specific real-time fluorescent PCR method established by the present invention is used to amplify, and the specificity of the method established by the present invention is detected. sex. Observe whether the amplification curve is generated. The experimental results are attached figure 1 shown. The experimental results showed that only J163 had exponential am...

Embodiment 3

[0066] Embodiment 3: Sensitivity test and standard curve establishment of detection system of the present invention

[0067] The initial template concentration of J163 genomic DNA was measured by ND2000C micro-spectrophotometer to be 200ng / μL. 100ng / μL, 10ng / μL, 1ng / μL, 100pg / μL, 10pg / μL, and 1pg / μL J163 genomic DNA templates were tested after TE dilution, and three replicates were set for each concentration. When more than 15pg (10pg / μL, 1.5μL) genomic DNA was used as a template, the amplification reaction showed a fluorescence increase, and the SD of the Ct values ​​measured in three parallels at each concentration ranged from 0.017 to 0.213, all less than 0.25. Data Table 1 shows. However, when 1.5pg (1pg / μL, 1.5μL) genomic DNA was used as a template, there was no positive amplification curve. It shows that the real-time fluorescent PCR system can detect the minimum detection limit of J163 genomic DNA as 15pg. Since the genomic DNA of alfalfa is estimated to be 1510Mbp, c...

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Abstract

The invention discloses a primer and a probe for the real-time fluorescent PCR detection of transgenic alfalfa J163 strains and a detection method and applications thereof. For transgenic alfalfa J163 strains which are provided by Monsanto Company in America and do not have the Security Certificate of Agricultural Genetically Modified Organisms awarded by the Ministry of Agriculture in China, the invention provides a pair of specific primer and probe for the first time, through the optimization of real-time fluorescent PCR detection systems, a specific real-time fluorescent PCR detection system for alfalfa J163 strains is successfully established, and the system is simple in operation, good in specificity and repeatability, accurate in detection results and high in stability, detects that the lowest DNA concentrations is 15 pg, and can quickly, accurately and steadily carry out detection analysis and authentication on the components of transgenic alfalfa J163 strains.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, to a specific real-time fluorescent PCR detection primer, probe, detection method and application of a transgenic alfalfa J163 strain. Background technique [0002] With the increase of my country's animal husbandry, especially dairy cows, the demand for plant feed, especially alfalfa, is also increasing every year. Alfalfa is the fourth largest crop in the United States after corn, wheat, and soybean. Because its protein content accounts for about 17% to 20% of the total hay mass, it is an excellent forage crop compared with other leguminous pastures. As of July this year, there were 71 American alfalfa manufacturers registered in my country. According to statistics, from January to June 2014, China imported a total of 405,600 tons of alfalfa, a year-on-year increase of 26.94%; the total import value was 149.6677 million US dollars, A year-on-year increase of 23.08%. Among them, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2561/113C12Q2561/101C12Q2531/113
Inventor 刘二龙卢丽吕英姿张旺蒋湘林惠娇郑高彬秦焯敏林学勤樊武疆姚柏辉王定国
Owner 黄埔出入境检验检疫局综合技术服务中心
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