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PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) assay primer and assay method for transgenic rapeseed RT73 strain

The technology of PCR-DHPLC and detection method is applied in the field of PCR-DHPLC detection primers of transgenic rapeseed RT73 lines, which can solve the problems such as no detection method, and achieve the effects of reliable detection method, high resolution and good expansion performance.

Inactive Publication Date: 2013-06-26
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-DHPLC detection method for the transgenic rapeseed RT73 line

Method used

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  • PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) assay primer and assay method for transgenic rapeseed RT73 strain
  • PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) assay primer and assay method for transgenic rapeseed RT73 strain
  • PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) assay primer and assay method for transgenic rapeseed RT73 strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1D

[0026] Example 1DNA Extraction

[0027] (1) DNA extraction: Refer to the method provided by the Plant Genomic DNA Extraction Kit (QIAGEN, Cat. No. 69104) to slightly improve the DNA extraction. The specific operation steps are as follows:

[0028] ① AP1 solution and Buffer AE preheated at 65°C;

[0029] ② After grinding the sample to powder with liquid nitrogen, take an appropriate amount of sample into a 1.5mL centrifuge tube;

[0030] ③Add 400 μL of preheated AP1 solution and 4 μL of 100 mg / mL RNase to the centrifuge tube, mix thoroughly, and put in a water bath at 65°C for 10 min-15 min, during which time shake and mix 2-3 times;

[0031] ④After the water bath is completed, add 130 μL AP2 solution directly, shake and mix well, and then ice bath for 5 minutes. Do not shake in the ice bath, and centrifuge at 14000r / min for 5 minutes;

[0032] ⑤Pipe the supernatant into the QIA shredder Mini spin column, that is, the purple column in the kit, centrifuge at 14000rpm / min for 2...

Embodiment 2D

[0045] Example 2 DNA concentration determination

[0046] 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:

[0047] DNA purity = OD260 / OD280

[0048] DNA concentration=50×OD260mg / mL

[0049] The purity ratio of DNA was between 1.7 and 1.9, and the concentration was greater than 10ng / μL.

Embodiment 3

[0050] Embodiment 3PCR amplification

[0051] Primers were designed according to the DNA sequence of the transgenic rapeseed RT73 strain (Table 1), and the DNA of the following samples was extracted for specific detection: transgenic rapeseed line MS1, transgenic rapeseed line RF1, transgenic rapeseed line RF2, transgenic rapeseed line RF3, transgenic rapeseed line RT73 , Transgenic rapeseed line MS8, genetically modified rapeseed line T45, genetically modified rapeseed line Topas19 / 2, non-genetically modified rapeseed, genetically modified corn line 3272, genetically modified corn line 59122, genetically modified corn line Bt11, genetically modified corn line Bt176, genetically modified corn line GA21, genetically modified corn line MIR604, GM corn line MON810, GM corn line MON863, GM corn line MON88017, GM corn line MON89034, GM corn line NK603, GM corn line TC1507, GM corn line T25, non-GM corn, non-GM corn, GM soybean line A2704- 12. Genetically modified soybean strain A55...

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Abstract

The invention discloses a PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) assay primer and assay method of transgenic rapeseed RT73 strain. The primer is strong in specificity, and can be used in PCR and used for specifically amplifying a DNA (deoxyribonucleic acid) fragment of the transgenic rapeseed RT73 strain, and the amplified product can be applied to subsequent DHPLC analysis. The assay method provides an assay method for the transgenic rapeseed RF2 strain, which is convenient to operate, good in extension performance and strong in specificity. The PCR amplified product is analyzed by utilizing the DHPLC, the resolution of the fragment of the PCR amplified can be a plurality of basic groups, and the resolution rate is high. The primer and assay method provided by the invention provide a simple, convenient, effective and reliable assay method to assay of the transgenic rapeseed RT73 strain, and are particularly applicable to port inspection and quarantine and other departments.

Description

technical field [0001] This application relates to the detection of transgenic products, in particular to a PCR-DHPLC detection primer and detection method of a transgenic rapeseed RT73 strain. Background technique [0002] Currently, conventional PCR, real-time fluorescent PCR, PCR-gene chip and other detection methods are mainly used for the detection of transgenic rapeseed. 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 sens...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11G01N30/02
Inventor 章桂明向才玉潘广凌杏园
Owner SHENZHEN AUDAQUE DATA TECH
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