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PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified rice strain KMD

A technology of PCR-DHPLC and transgenic rice, which is applied in the direction of biochemical equipment and methods, recombinant DNA technology, microbial determination/inspection, etc., to achieve the effect of reliable detection method, good expansion performance, high sensitivity and high sensitivity

Inactive Publication Date: 2013-03-06
SHENZHEN AUDAQUE DATA TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no PCR combined with DHPLC detection technology (PCR-DHPLC) for the transgenic rice line Kemo rice

Method used

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  • PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified rice strain KMD
  • PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified rice strain KMD
  • PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified rice strain KMD

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 DNA Extraction

[0031] Sample DNA was extracted by CTAB method, as follows:

[0032] a) Weigh 5 g of the sample, add liquid nitrogen to the mortar and grind until the sample is a powder with a size of about 0.5 mm;

[0033] b) Weigh 300 mg of the ground sample, quickly transfer it to a 2 mL centrifuge tube, add 700 μL of CTAB extract solution preheated at 65 °C, mix well, and put it in a water bath at 65 °C for 30 min;

[0034] c) Add 5 μL RNase (10 mg / mL), and bathe in water at 37°C for 30 minutes;

[0035] d) Add an equal volume of Tris saturated phenol, mix thoroughly, and centrifuge at 12000r / min for 15min;

[0036] e) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24:1) to mix, and centrifuge at 12000r / min for 15min;

[0037] f) Take the supernatant, add an equal volume of chloroform / isoamyl alcohol (24:1) to mix, and centrifuge at 12000r / min for 15min;

[0038]g) Add an equal volume of pre-cooled isopropanol, shake gently, ...

Embodiment 2

[0041] Example 2 DNA concentration determination

[0042] The concentration and purity of the extracted sample DNA were measured; the absorbance values ​​at 260nm and 280nm were measured by an ultraviolet spectrophotometer, and the purity and concentration of nucleic acid were calculated respectively. The calculation formula is as follows:

[0043] DNA purity = OD260 / OD280

[0044] DNA concentration=50×OD260mg / mL

[0045] The purity ratio of DNA was between 1.7 and 1.9, and the concentration was greater than 10ng / μL.

Embodiment 3

[0046] Embodiment 3 PCR amplification

[0047] Design the binding site of the specific detection primer according to the transgenic rice line Kechi rice, and add the regulatory sequence at the 5' end of the specific detection primer binding site, synthesize the detection primer (Table 1) containing the regulatory sequence, and use the method of adding the regulatory sequence The upstream / downstream primers were used for PCR amplification, and the sample settings included: non-transgenic rice DNA, Bt63DNA, genetically modified rice line Kemodao 1DNA, genetically modified rice line Kefeng 6 DNA, MON88017DNA, genetically modified corn line Bt176DNA, genetically modified corn Bt11DNA, Corn MON863 DNA, soybean line A2704-12 DNA, soybean line A5547-12 DNA, non-transgenic canola DNA, LLcotton25 DNA, non-transgenic cotton DNA, potato EH92-527-1 DNA, and a water control.

[0048] Table 1 The detection primer binding sites and primers of the transgenic rice line Kemo rice

[0049]

...

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Abstract

The invention discloses a PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and a detection method for genetically modified rice strain KMD. The primer has strong specificity and can be used for PCR amplification and DHPLC analysis. The detection method is simple and convenient to operate, good in expansion performance and high in sensitivity. The DHPLC is used to analyze PCR amplified products, fragment size resolution can reach multiple bases, and resolution ratio is high. The PCR-DHPLC detection primer and the detection method for genetically modified rice strain KMD have the advantages that the detection method is simple, convenient, effective, reliable and especially suitable for departments of port inspection and quarantine and the like.

Description

technical field [0001] The invention relates to the detection of a transgenic product, in particular to a PCR-DHPLC detection primer and a detection method of a transgenic rice strain. Background technique [0002] At present, the detection methods of transgenic rice mainly adopt conventional PCR, real-time fluorescent PCR, PCR-gene chip and other detection methods. The traditional conventional PCR detection method has certain limitations in terms of platform expansion. With the increase of targets to be detected, it is necessary to re-optimize the amount and ratio of each set of primers in the system, and take into account factors such as amplification efficiency and workload. On the other hand, the commonly used gel electrophoresis method to analyze the amplification products has low discrimination efficiency and unsatisfactory detection results. Although real-time fluorescent PCR has advantages over conventional PCR detection methods in terms of detection sensitivity, du...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 章桂明向才玉凌杏园潘广程颖慧康林李鹤遥
Owner SHENZHEN AUDAQUE DATA TECH
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