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Primer for PCR identification of kidney bean and PCR identification method

A combination technology of kidney beans and primers, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve problems such as poor repeatability, non-specific amplification, and low annealing temperature, and achieve accurate results, The effect of quick operation and shortened test time

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

AI Technical Summary

Problems solved by technology

However, this method has the following disadvantages: Since short primers of about 10 base pairs are used, the annealing temperature is low, and non-specific amplification is easily caused by annealing misoperation
Moreover, PCR using RAPD markers includes specific amplified fragments and redundant amplified fragments required for identification, resulting in multiple PCR products, so the repeatability is poor, and skilled technology is required for identification

Method used

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  • Primer for PCR identification of kidney bean and PCR identification method
  • Primer for PCR identification of kidney bean and PCR identification method

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Experimental program
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Effect test

Embodiment 1

[0036] The design of embodiment 1 primer

[0037]According to the vegetable trnL sequence, use software and Primer 3 to design primer combination I, the primer sequence is:

[0038] Forward: 5'-GAATCCTTTCACCAAAATTCCA-3'

[0039] Reverse: 5'-TTTCAACTTCGAGTTGGTTTGA-3'.

[0040] According to the trnL sequence of kidney bean, the primer combination II was designed using software and Premier 5, and the primer sequence is:

[0041] Forward: 5'-TAACAAACGAAATTGACG-3'

[0042] Reverse: 5'-TCCGATTATGATAAAGTGAA-3'.

Embodiment 2

[0043] The extraction of embodiment 2 total DNA

[0044] (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.

[0045] (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 .

[0046] (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.

[0047] (4) Draw the supernatant and place it in another sterilized 1.5mL centrifuge tube.

[0048] (5) Add an equal volume of chloroform / isoamyl alcohol (24:1) and repeat steps (3) and (4).

[0049] (6) Add an equal or double volume of absolute ethanol (pre-cooled at -2...

Embodiment 3

[0053] The establishment of embodiment 3PCR amplification method

[0054] 1. PCR reaction system

[0055] 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, 25mM MgCL 2 2μL, 10mM dNTPs 2μL, 10μM Primers 2μL, 5U / μL Taq DNApolymerase 0.1μL, ddH 2 O 15.4 μL.

[0056] 2. PCR reaction conditions

[0057] After putting the sample tube into the ABI PCR instrument, set the following conditions for the reaction according to the primer combination I: pre-denaturation at 94°C for 3 min, then denaturation at 94°C for 30 sec, annealing at 66°C for 30 sec, extension at 72°C for 1 min, 35 cycles, and finally 72°C Extend for 5 minutes; set the following conditions for the reaction according to primer combination II: pre-denaturation at 94°C for 3 minutes, then denaturation at 94°C for 30 sec, annealing at 48°C for 30 sec, extension at 72°C for 1 min, 35 cycles, and finally e...

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Abstract

The invention provides a primer for PCR identification of kidney beans and a PCR identification method, wherein the primer can amplify chloroplast trnL genes of the kidney beans to generate specific amplified fragments of the kidney beans, and concrete nucleotide sequences are shown as sequence tables SEQ ID No. 1&2 and No. 3&4. The PCR identification method of the kidney beans comprises the following steps: utilizing the primer for PCR amplification by taking the total DNA of a sample as a template, and judging results according to agarose electrophoresis after reaction is finished. The primer has good specificity, high accuracy and good sensitivity, and a rapid and simple detection method is provided for identifying the species resource of the kidney beans.

Description

technical field [0001] The invention relates to biological detection and identification technology, in particular to primers for PCR identification of kidney bean biological resources and a PCR identification method using the primers. Background technique [0002] Phaseolus vulgaris, also known as green beans, kidney beans, and jade beans, is a crop with high protein, low fat, and medium starch content. It is a very important source of plant protein and has high edible value. In addition, it is also a high nutritional value. feed. The medicinal value of kidney bean is also very high. Its seeds can be used as medicine, and its nature and taste are sweet and flat. Kidney bean pods and seeds also contain phytohemagglutinin, which is a kind of glycoprotein, which can agglutinate human cells, stimulate embryonic transformation of lymphocytes, inhibit the transfer of white blood cells and lymphocytes, and can improve chemical Efficacy of therapy and radiation therapy. With my c...

Claims

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Application Information

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