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Method for rapid detection of transgenic rice line TT51-1

A transgenic rice, rapid technology, applied in the field of molecular biology, can solve the problems of unsuitable rapid detection, poor mobility, complicated operation process, etc., and achieve the effect of fast and efficient detection method, simple operation and high sensitivity

Inactive Publication Date: 2014-05-07
TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the PCR detection method is mature and stable, the reaction system and operation process are relatively complicated, requiring professionals; the PCR amplification instrument is expensive, has high requirements for the detection environment and poor mobility, and is not suitable for rapid on-site detection; qualitative PCR amplification reaction and Electrophoretic detection takes a long time, and the commonly used dye EB is a strong carcinogen; quantitative PCR instruments and consumables are expensive, and need to be connected to a computer to monitor the test results in real time; the above factors limit its application as a rapid on-site detection

Method used

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  • Method for rapid detection of transgenic rice line TT51-1
  • Method for rapid detection of transgenic rice line TT51-1
  • Method for rapid detection of transgenic rice line TT51-1

Examples

Experimental program
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Effect test

Embodiment 1

[0060] (1) Reagents: BstDNA polymerase large fragment produced by BioLabs (NEWENGLAND) and 10 times Thermal Buffer solution; TT51-1 specific primer mixture; calcein solution, manganese chloride solution; magnesium sulfate solution; dNTPs; BSA; ionized water. DNA templates include negative control, positive control, negative sample 1, negative sample 2, positive sample 1, and positive sample 2.

[0061] (2) Amplification reaction system: the total volume of the amplification reaction is 25 μL, and its various components and dosages are: 10×Buffer 2.5 μL, 10 mM dNTPs 2 μL, 50 mM MgSO 4 2 μL, 12.5×BSA 2 μL, 625 μM Calcein 2 μL, 12.5 mMMnCl 2 1 μL, primer mixed solution 1 μL, 8000U / ml Bst DNA polymerase large fragment 1 μL, template DNA 1 μL, make up to 25 μL with sterilized deionized water, mix well and centrifuge;

[0062] (3) Amplification reaction procedure: incubate at 61°C for 60 minutes, then keep at 80°C for 2 minutes, and store at 4°C;

[0063] (4) After the amplificat...

Embodiment 2

[0065] (1) Reagents: Bst DNA polymerase large fragment produced by BioLabs (NEW ENGLAND) and 10 times Thermal Buffer solution; TT51-1 specific primer mixture; calcein solution, manganese chloride solution; magnesium sulfate solution; dNTPs; BSA ;Deionized water. The DNA template includes a negative control, a positive control, a test sample 1, a test sample 2, a test sample 3, and a test sample 4.

[0066] (2) Amplification reaction system: the total volume of the amplification reaction is 25 μL, and its various components and dosages are: 10×Buffer 2.5 μL, 10 mM dNTPs 2 μL, 50 mM MgSO 4 2 μL, 12.5×BSA 2 μL, 625 μM Calcein 2 μL, 12.5 mMMnCl 2 1 μL, primer mixture solution 1 μL, 8000U / ml BstDNA polymerase large fragment 1 μL, template DNA 2 μL, make up to 25 μL with sterilized deionized water, mix well and centrifuge;

[0067] (3) Amplification reaction procedure: incubate at 61°C for 60 minutes, then keep at 80°C for 2 minutes, and store at 4°C;

[0068] (4) After the amp...

Embodiment 3

[0070] Comparative experiment: the comparison between the current assay method of transgenic rice TT51-1 and the assay method of the present invention.

[0071] (1) Reagents for this method: Bst DNA polymerase large fragment and 10 times Buffer solution produced by BioLabs (NEW ENGLAND); TT51-1 specific primer mixture; calcein solution, manganese chloride solution; magnesium sulfate solution; dNTPs; BSA; deionized water. The DNA templates include 10ng (about 20000 copies), 1ng (about 2000 copies), 100pg (about 200 copies), 50pg (about 100 copies) and 10pg (about 20 copies) of transgenic rice TT51-1 components. 5pg (about 10 copies), 1pg (about 2 copies) samples.

[0072] (2) Amplification reaction system of this method: the total volume of the amplification reaction is 25 μL, and its various components and dosages are: 10×Buffer 2.5 μL, 10mM dNTPs 2 μL, 50mM MgSO 4 2 μL, 12.5×BSA 2 μL, 625 μM Calcein 2 μL, 12.5 mM MnCl 21 μL, primer mixed solution 1 μL, 8000U / ml Bst DNA po...

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Abstract

The invention discloses a method for rapid detection of transgenic rice line TT51-1 and primers for use. The method belongs to the technical field of molecular biology, and relates to detection methods of transgenic products, the principle is as follows: using 6 specific primers and a DNA polymerase with chain displacement activity for amplification of a sample template DNA at 61 DEG C, amplified products can reach 109-1010 copies in a short period of time, result identification can be realized as follows: determining whether the DNA is amplified or not by direct observation of color change in a reaction tube by naked eyes, and the method has the characteristics of being high in efficiency, fast, simple in operation, high in specificity and the like, and provides a convenient means for the rapid detection of transgenic rice.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and relates to a detection method for a transgenic product, specifically a method for rapidly detecting a transgenic rice strain TT51-1, which can be judged by observing the color change of the reaction tube with the naked eye or observing the agarose gel electrophoresis. Whether it contains the ingredients of transgenic rice TT51-1. Background technique [0002] Since the first commercial GM crop was approved for marketing in 1994, the application and development of GM technology in the global agricultural field has been growing steadily. According to statistics, the global planting area of ​​genetically modified crops has continued to grow for 16 consecutive years. In 2011, the global planting area of ​​genetically modified crops reached 160 million hectares, 94 times that of 1996, accounting for about 10% of the global available arable land. During the 15 years from 1996 to 2011, 29...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q2531/119
Inventor 陈锐王永兰青阔赵新朱珠郭永泽
Owner TIANJIN INSTITUE OF QUALITY STANDARD & TESTING OF AGRICULTUAL PRODS
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