Kit and method for detection of transgenic maize Mon89034 and derivative varieties thereof through gene amplification at constant temperature

A technology of constant temperature gene amplification and transgenic corn, applied in the field of molecular biology, to achieve the effects of high sensitivity, high specificity and easy identification

Inactive Publication Date: 2012-11-14
广州迪澳生物科技有限公司
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AI Technical Summary

Problems solved by technology

[0009] Loop-Mediated Isothermal Amplification Technology (Loop-Mediated Isothermal Amplification, hereinafter referred to as LAMP method) is a gene amplification technology developed by Japan's Eiken Chemical Co., Ltd. around 2000. It is fast, simple, accurate, easy to popularize, With the advantages of safety and reliability, there is currently no kit that applies the LAMP method to the rapid detection of transgenic corn Mon89034 and its derivatives

Method used

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  • Kit and method for detection of transgenic maize Mon89034 and derivative varieties thereof through gene amplification at constant temperature

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1 contains the kit of chromogen and detection method thereof:

[0051] A kit for rapid detection of transgenic maize Mon89034 and its derivatives by constant temperature gene amplification was prepared according to the following formula:

[0052] (1) Primer mixture: Prepare synthetic primer dry powder into a mother solution with a concentration of 100 μM, then take 1 μl each of outer primer 1 and outer primer 2, and 8 μl each of inner primer 1 and inner primer 2, and mix them thoroughly. The sequences of the primers are respectively :

[0053] Outer primer 1: CTATGTAAGAGGTGTTTGAA; as shown in SEQ ID No.1;

[0054] Outer primer 2: GTAAGATGTGAGTATGATCC; as shown in SEQ ID No.2;

[0055] Internal primer 1: GTCCATCTATCAAATCAGCACCGTCTATCCACGGTCCGTGCGCACC; as shown in SEQ ID No.3;

[0056] Internal primer 2: GAACAACATCTCTGGAGTCGGTGAGTCCGGCGTACTGCGCCACC; as shown in SEQ ID No.4;

[0057] (2) DNA polymerase: Bst DNA polymerase, the concentration is 8U / μl;

[005...

Embodiment 2

[0066] Embodiment 2 does not contain the kit of chromogenic agent and detection method thereof:

[0067] The kit is the same as Example 1 except that it lacks the chromogen in Example 1.

[0068] Detection method is except analyzing and judging reaction product result step, all the other are with embodiment 1, and the method for analyzing and judging result is:

[0069] Place the reaction tube in the turbidimeter and react according to the step (2). Observe the turbidity change of the precipitate in the reaction tube to judge the amplification result. If there is precipitation, it is positive, and if there is no precipitation, it is negative.

[0070] In this example, precipitation appeared in the PCR tube, indicating that the corn seeds to be tested contain or all of them are transgenic corn Mon89034 and its derivative varieties, and contain components of transgenic corn Mon89034.

Embodiment 3

[0071] Embodiment 3PCR reaction and the comparison of detection method sensitivity of the present invention:

[0072] A kit for rapid detection of transgenic maize Mon89034 and its derivatives by constant temperature gene amplification was prepared according to the following formula:

[0073] (1) Primer mixture: Prepare synthetic primer dry powder into a mother solution with a concentration of 100 μM, then take 1 μl each of outer primer 1 and outer primer 2, and 8 μl each of inner primer 1 and inner primer 2, and mix them thoroughly. The sequences of the primers are respectively :

[0074] Outer primer 1: CTATGTAAGAGGTGTTTGAA; as shown in SEQ ID No.1;

[0075] Outer primer 2: GTAAGATGTGAGTATGATCC; as shown in SEQ ID No.2;

[0076] Internal primer 1: GTCCATCTATCAAATCAGCACCGTCTATCCACGGTCCGTGCGCACC; as shown in SEQ ID No.3;

[0077] Internal primer 2: GAACAACATCTCTGGAGTCGGTGAGTCCGGCGTACTGCGCCACC; as shown in SEQ ID No.4;

[0078] (2) DNA polymerase: Bst DNA polymerase, other ...

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Abstract

The invention discloses a kit and a method for detection of transgenic maize Mon89034 and derivative varieties thereof through gene amplification at a constant temperature. The kit mainly comprises four primers, DNA polymerase, a reaction solution, a positive contrast and a negative contrast, wherein all the above-mentioned liquid are respectively disposed in a container; the kit also comprises a color-developing agent. The detection method is as follows: DNAs of a maize variety to be detected are extracted, the four specific primers and the DNA polymerase having strand displacement activity are used for amplification of a sample DNA template at a temperature of 63 to 65 DEG C, and amplification efficiency reaches 109 to 1010 copies in a short period of time; for identification of the maize variety to be detected, SYBR Green I is added and changes of colors are observed, or changes of turbidity of deposition in a reaction tube are observed by using a turbidity meter so as to determine whether amplification occurs or not, and then detection is carried out on the maize variety to be detected so as to determine whether the maize variety contains or is transgenic maize Mon89034 and a derivative variety thereof. The beneficial effects of speed, high efficiency, simple operation, high specificity, high sensitivity, easy identification, suitability for on-site detection and the like are obtained in the invention.

Description

【Technical field】 [0001] The invention belongs to the technical field of molecular biology, and relates to a detection kit for transgenic plant products and a detection method thereof, in particular to a kit for rapidly detecting transgenic corn Mon89034 and derivatives thereof based on a loop-mediated isothermal gene amplification technology and its detection method. 【Background technique】 [0002] According to the report "2010 Global Biotech / GM Crops Commercialization Development Status" recently published by the International Agricultural Biotechnology Application Service, about 100 million farmers have made planting decisions in the past 15 years due to the huge benefits brought by genetically modified crops . In the 15 years since GM crops were commercially planted, the cumulative area of ​​GM crops in the world has exceeded 1 billion hectares. In 2010, 15.4 million farmers in 29 countries around the world planted a total of 148 million hectares of biotech crops. Fro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 石磊李志勇高东微高苏娟叶宇鑫杨坚袁瑛娜
Owner 广州迪澳生物科技有限公司
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