PCR authenticating primer and method of oryza punctata

A wild rice, spot technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve problems such as research on unseen molecular detection methods, and avoid time-consuming and omissions, results Accurate and fast operation effect

Inactive Publication Date: 2010-05-05
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0004] At present, the identification of O.punctata at home and abroad is l...

Method used

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  • PCR authenticating primer and method of oryza punctata

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Embodiment 1

[0017] The design of embodiment 1 primer

[0018] According to the psbA-trnH sequence of Oryza spectabilis, primers were designed using software and Primer 3. After several rounds of screening, it was unexpectedly found that the specificity of the following primers was significantly better than other primers. The primer sequence is:

[0019] Forward: 5'-CGGCATTTCACGAGTTATGA-3'

[0020] Reverse: 5'-TTGCTTCAGCGAGATATTGG-3'.

Embodiment 2

[0021] The extraction of embodiment 2 total DNA

[0022] (1) Take 0.2-0.3g of plant tissue, preserve it with silica gel, cut it into pieces, put it in a mortar, add liquid nitrogen, grind it into a powder, transfer it to a sterilized 1.5mL centrifuge tube, generally add to 1 / 3 of the centrifuge tube volume.

[0023] (2) Add 500μLCTAB extract (20g / LCTAB, 1.4M NaCl, 0.1M Tris-HCl, 20mM EDTA, pH 8.0) preheated at 65°C to the centrifuge tube, mix well, and place in a 65°C water bath for 30 minutes .

[0024] (3) Take the sample out of the water bath, add chloroform / isoamyl alcohol (24:1) equal to the volume of the extract, mix well by inverting the centrifuge tube, and centrifuge at 12,000 rpm for 15 minutes.

[0025] (4) Draw the supernatant and place it in another sterilized 1.5mL centrifuge tube.

[0026] (5) Add an equal volume of chloroform / isoamyl alcohol (24:1) and repeat steps (3) and (4).

[0027] (6) Add an equal or double volume of absolute ethanol (pre-cooled at -2...

Embodiment 3

[0031] The establishment of embodiment 3PCR amplification method

[0032] 1. PCR reaction system

[0033] Use the total DNA as a template to carry out PCR reaction. There are samples in the 25μL reaction system: sample DNA 1μL (10-50ng), 10×PCR buffer (Mg 2+ free) 2.5μL, 25mMMgCL2 2μL, 10mM dNTPs 2μL, 10μM Primers 1μL / 1μL, 5U / μL TaqDNA polymerase 0.1μL, ddH 2 O 15.4 μL.

[0034] 2. PCR reaction conditions

[0035] After putting the sample tube into the ABI 7900 PCR instrument, set the following conditions for reaction: pre-denaturation at 94°C for 4 minutes, then denaturation at 94°C for 1 minute, annealing at 62°C for 1 minute, extension at 72°C for 1.5 minutes, 30 cycles, and finally extension at 72°C for 7 minutes. After the reaction, the amplified products were judged by agarose gel electrophoresis.

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Abstract

The invention provides PCR authenticating primer and method of oryza punctata. The nucleotide sequence of the primer is shown as the sequence table SEQ ID No. 1 and 2. The invention also provides a PCR detection method of the oryza punctata, and the detection method comprises the steps of carrying out PCR amplification by using a sample total DNA as a template through the primer and determining a result according to agaroseelectrophoresis after the reaction is ended. The primer has good specificity, and the detection method is quick and simple and has high accuracy and good sensitivity. The invention provides an effective detection method for the authentication of the oryza punctata.

Description

technical field [0001] The invention relates to biological detection technology, in particular to a primer and a PCR detection method for detecting biological resources of spotted wild rice. Background technique [0002] Due to the long-term natural selection of various disasters and adverse environments, wild rice contains a large number of beneficial genes such as disease resistance, insect resistance, stress resistance and male sterility. important gene source. With the development of industry in the past ten years, the process of urbanization, environmental pollution, population growth and other human factors have brought serious harm to the survival of wild rice, coupled with the intensification of species loss, the distribution area of ​​the main wild rice is decreasing Therefore, it is particularly important to protect and identify wild rice. Although the basic framework of the origin and phylogenetic relationship of Oryza polyploids has been formed, the classificat...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 徐涛许瑾
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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