Method for testing multi-PRC reaction of transgenic fruit

A detection method and genetically modified technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of difficult detection of genetically modified fruits, false positives, etc., and achieve the effect of obvious superiority and simple method

Inactive Publication Date: 2009-11-11
陈军 +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] The main purpose of the present invention is to provide a multiplex PCR detection method for transgenic fruit, which solves the problems in the prior art that transgenic fruit is difficult to detect or that it is easy to cause false positives through commonly used PCR methods.

Method used

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  • Method for testing multi-PRC reaction of transgenic fruit
  • Method for testing multi-PRC reaction of transgenic fruit
  • Method for testing multi-PRC reaction of transgenic fruit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1 Double PCR detection and verification of transgenic papaya

[0099] 1. Synthesis of PCR primers

[0100] The PCR primer sequence of the present invention is a synthetic primer sequence; the primer sequence information is as follows:

[0101] Table 1 Primer sequence information table

[0102]

[0103] 2. Extraction of sample DNA

[0104] Instruments and equipment: Bio-rad PCR thermal cycler; Bio-rad electrophoresis instrument and gel imaging system; UV-visible spectrophotometer; micropipette; microcentrifuge and refrigerated centrifuge; Drying equipment; various commonly used glassware.

[0105] Material preparation:

[0106] Pre-extraction buffer: 50mmol / L Tris·HCl (pH 8.0), 0.7mol / L NaCl, 10mmol / L EDTA (pH 8.0).

[0107] CTAB extraction buffer: add 2% (m / V) CTAB, 2% PVP, 20mL / L β-mercaptoethanol (add before use) to the pre-extraction buffer.

[0108] Chloroform / isoamyl alcohol (24:1; 70% ethanol; 1×TE buffer: 10mmol / L Tris·HCl (pH 8.0), 1mmol / L EDTA ...

Embodiment 2

[0137] Example 2 detects the triple PCR of Hainan Hetai papaya and Wangpai papaya

[0138] 1. Synthesis of PCR primers

[0139] The PCR primer sequence of the present invention is a synthetic primer sequence; the primer sequence information is as follows:

[0140] Table 2 Primer sequence information table

[0141]

[0142] 2. Extraction of sample DNA

[0143] Experimental apparatus and experimental reagent are the same as embodiment 1. The DNA extracted from the tender leaves of evergreen trees was used as a negative control, and ddH 2 O is the blank control, and the transgenic papaya DNA is used as the positive control to test the two products of Hainan Hetai papaya and Wangpai papaya.

[0144] The experimental procedure is similar to Example 1, wherein the CTAB extraction buffer and β-mercaptoethanol for DNA extraction are preheated at 70°C. During the PCR amplification, three primer pairs were used to amplify the 35S promoter, the NOS terminator, and the plant endog...

Embodiment 3

[0149] Example 3 Triple PCR Detection of Laoxie Brand Papaya, Hami Melon, Banana and Pear

[0150] Select Laoxie brand papaya, cantaloupe, banana, and pear as the test samples for detection, and the steps are similar to Example 1. The DNA extracted from the tender leaves of evergreen trees is used as the negative control, and ddH 2 O is a blank control, and transgenic papaya DNA is used as a positive control to detect four products of Laoxie papaya, Hami melon, banana and pear. The CTAB extraction buffer and β-mercaptoethanol for DNA extraction were preheated at 60°C. During the PCR amplification, three primer pairs were used to amplify the 35S promoter, the NOS terminator, and the plant endogenous gene rbcL respectively. The primer sequence information is as follows:

[0151] Table 3 Primer sequence information table

[0152]

[0153] In the triple PCR reaction system, 2.5 μL of 10× amplification buffer, final dNTP concentration of 0.4 mM, final concentration of primers...

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Abstract

The invention discloses a method for testing the multi-PRC reaction of a transgenic fruit. The method is characterized by including the following steps: (1) proper primer sequences are designed and selected; (2) the hexadecyl triethyl ammonium bromide method is used for extracting the genome DNA of a fruit to test and for determining the concentration of DNA; in the primer sequence of the step (1), the extracted DNA is used as a template for the multi-PCR amplification of 35S promoter, NOS terminator, plant endogenous rbcl gene; and (3) restriction enzyme is used for the restriction endonuclease reaction of the result of the multi-PCR amplification, and the agarose gel electrophoresis analysis and the DNA sequencing are used for judging whether the fruit contains transgenic ingredients or not. The method can successfully distinguish a transgenic fruit from a non-transgenic fruit, and can quickly, conveniently and accurately determine the transgenic ingredients in the fruits.

Description

technical field [0001] The invention belongs to the technical field of transgenic fruit safety and detection, and in particular relates to a multiplex PCR detection method for transgenic fruit; the invention also relates to a kit for detecting transgenic fruit. Background technique [0002] Introduce foreign genes into other specific organisms through genetic engineering technology to cause positive genetic mutations, transform the genetic material of the organisms, and transform them to the goals that people need in terms of shape, nutritional quality, and consumption quality. , becoming a new type of organism with entirely new properties. The organisms are called genetically modified organisms or genetically modified organisms (Genetically Modified Organisms, GMOs), including transgenic animals, plants and microorganisms. Foods that are directly eaten by genetically modified organisms or produced as processed raw materials are collectively referred to as "Genetically Modi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 陈军肖琳戴岚陈骏锋朱振华
Owner 陈军
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