PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain CBH351

A PCR-DHPLC, CBH351 technology, applied in biochemical equipment and methods, DNA/RNA fragments, measuring devices, etc., to achieve the effects of high sensitivity and resolution, good scalability, and reliable detection methods

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

Currently, there is no PCR-DHPLC detection technique (PCR-DHPLC) for transgenic maize line CBH351

Method used

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  • PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain CBH351
  • PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain CBH351
  • PCR-DHPLC (polymerase chain reaction-denaturing high performance liquid chromatography) detection primer and detection method for genetically modified maize strain CBH351

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 DNA extraction

[0031] Use CTAB method to extract sample DNA, as follows:

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

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

[0034] c) Add 5μL of RNase (10mg / mL), 37℃ water bath for 30min;

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

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

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

[0038] g) Add an equal volume of pre-cooled isopropanol, shake it gently, place it in a refrigerator at -20°C for 30 minutes...

Embodiment 2

[0041] Example 2 DNA concentration determination

[0042] Determine the concentration and purity of the extracted sample DNA; use an ultraviolet spectrophotometer to measure the absorbance at 260nm and 280nm, and calculate the purity and concentration of nucleic acid respectively. The calculation formula is as follows:

[0043] DNA purity=OD260 / OD280

[0044] DNA concentration=50×OD260mg / mL

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

Embodiment 3

[0046] Example 3 PCR amplification

[0047] According to the genetically modified corn CBH351 strain, the binding site of the specific detection primer was designed, and a regulatory sequence was added to the 5'end of the binding site of the specific detection primer, and the detection primer containing the regulatory sequence was synthesized (Table 1). / Downstream primers for PCR amplification, sample settings include: non-transgenic maize DNA, maize line MON88017 DNA, maize line MIR604 DNA, maize line Bt176 DNA, maize line CBH351 DNA, maize line Bt11, maize line MON810 DNA of corn line GA21, DNA of corn line NK603, DNA of corn line MON863, DNA of corn line MON89034, DNA of corn line T25, DNA of soybean line A2704-12, DNA of soybean line A5547-12, non DNA from genetically modified rapeseed, DNA from LLcotton25 strain, DNA from non-transgenic cotton, DNA from Bt63 strain, DNA from potato strain EH92-527-1.

[0048] Table 1 Detection of primer binding sites and primers for transge...

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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 maize strain CBH351. 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 maize strain CBH351 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 a detection of genetically modified products, in particular to a PCR-DHPLC detection primer and a detection method of genetically modified corn strains. Background technique [0002] At present, the detection methods of genetically modified corn mainly adopt conventional PCR, real-time fluorescent PCR, PCR-gene chip and other detection methods. Traditional conventional PCR detection methods have certain limitations in terms of platform expansion. As the targets to be tested increase, the amount and ratio of each set of primers in the system need to be re-optimized, and factors such as amplification efficiency and workload need to be re-optimized. Larger; on the other hand, the commonly used method of gel electrophoresis to analyze the amplified products has low discrimination efficiency and unsatisfactory detection results. Although real-time fluorescent PCR has more advantages in detection sensitivity and other aspects of conventi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11G01N30/02
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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