Nested-PCR (polymerase chain reaction) amplification detection system and application of citrus yellow shoot Candidatus Liberobacter asiaticus

A technology of citrus huanglongbing and amplification system, applied in the field of molecular biology, can solve problems such as high energy consumption, and achieve the effects of low cost, small amount of reagents, and pollution reduction

Inactive Publication Date: 2014-05-28
广西壮族自治区农业科学院园艺研究所 +1
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, in the process of detecting citrus Huanglongbing by Nest’s PCR technology, although the final detection result can be easily achieved with a high dilution factor, the higher the dilution factor, the more RNase-free water or 0.1% DEPC working solution is required , which means more energy consumption

Method used

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  • Nested-PCR (polymerase chain reaction) amplification detection system and application of citrus yellow shoot Candidatus Liberobacter asiaticus
  • Nested-PCR (polymerase chain reaction) amplification detection system and application of citrus yellow shoot Candidatus Liberobacter asiaticus
  • Nested-PCR (polymerase chain reaction) amplification detection system and application of citrus yellow shoot Candidatus Liberobacter asiaticus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1. Experimental materials

[0024] 1. Plant material

[0025] The leaf materials of 7 Shatangju suspected of Huanglongbing, all collected from Guangxi, were numbered AT5-2, AT5-4, AT5-49, AT5-55, AT5-71, AT5-84, AT5-141, among them, AT5- 2. AT5-4 was collected from Wuming County, AT5-49 and AT5-55 were collected from Cenxi County, AT5-71 was collected from Fangchenggang City, AT5-84 was collected from Long’an County, and AT5-141 was collected from Xilin County.

[0026] 2. Primers

[0027] According to the 16S rDNA sequence of Candidantus Liberibacter asiaticus, primers fd1 / fd2 (SEQ ID NO:1 / SEQ ID NO:2), OI1 / OI2 (SEQ ID NO:3 / SEQ ID NO:4) were designed ), the length of the target nucleic acid fragment is 1160bp, synthesized by Shanghai Jierui Bioengineering Co., Ltd., the primer sequence is as follows:

[0028] fd 1 : 5′-AGAGTTTGATCCTGGCTCAG-3;

[0029] fd 2 : 5'-AAGGAGGTGATCCAGCC-3'.

[0030] OI 1 : 5'-GCGCGTATGCAATACGAGCGGCA-3';

[0031] OI 2 : 5'-GCCTCGCGACT...

Embodiment 2

[0055] The 7 DNA extracts prepared in Example 1 were diluted 100 times in volume to prepare DNA templates with a concentration of 2.75 ng / μl for use.

[0056] Using 16S rDNA Primer fd of Candidantus Liberibacter asiaticus 1 / fd 2 , OI 1 / OI 2 PCR amplification detection was performed on 7 DNA samples. The first round of PCR amplification system: 1.0 μl DNA template (concentration: 2.75ng / μl), 5 μl 2×Taq PCR MasterMix, 0.5 μlfd 1 / fd 2 Primer, add ddH 2 O was added to 15 μl, and the 2×Taq PCR Master Mix did not contain dyes, and the concentrations of dATP, dCTP, dGTP, and dTTP were all 0.4 mmol / L; Mgcl 2 The concentration is 4mmol / L; the total concentration of Taq enzyme and Pfu DNA polymerase is 0.05U / μL, and the primer fd 1 , primer fd 2 The concentration is 5 μmol / L, and the volume is 0.25 μl. The first round of PCR amplification reaction program: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 sec, annealing at 58°C for 45 sec, extension at 72°C for...

Embodiment 3

[0059] The 7 DNA extracts prepared in Example 1 were diluted 100 times in volume to prepare DNA templates with a concentration of 2.75 ng / μl for use.

[0060] Using 16S rDNA Primer fd of Candidantus Liberibacter asiaticus 1 / fd 2 , OI 1 / OI 2 PCR amplification detection was performed on 7 DNA samples. The first round of PCR amplification system: 1.0 μl DNA template (concentration: 2.75ng / μl), 5 μl 2×Taq PCR MasterMix, 0.5 μlfd 1 / fd 2 Primer, add ddH 2 O was supplemented to 20 μl, and the 2×Taq PCR Master Mix did not contain dyes, and the concentrations of dATP, dCTP, dGTP, and dTTP were all 0.4 mmol / L; Mgcl 2 The concentration is 4mmol / L; the total concentration of Taq enzyme and Pfu DNA polymerase is 0.05U / μL, and the primer fd 1 , primer fd 2 The concentration is 5 μmol / L, and the volume is 0.25 μl. The first round of PCR amplification reaction program: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 sec, annealing at 58°C for 45 sec, extension at 7...

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Abstract

The invention relates to PCR (polymerase chain reaction) detection of citrus yellow shoot Candidatus Liberobacter asiaticus, particularly relates to a nested-PCR detection system and application of citrus yellow shoot Candidatus Liberobacter asiaticus, and belongs to the field of molecular biology. The provided nested-PCR amplification detection system of citrus yellow shoot Candidatus Liberobacter asiaticus comprises a first-round PCR amplification system and a second-round PCR amplification system, final volumes of the first-round PCR amplification system and the second-round PCR amplification system range from 10 mu L to 20 mu L, and each of the first-round PCR amplification system and the second-round PCR amplification system comprises 1.0 mu L of DNA (deoxyribonucleic acid) templates, 5 mu L of 2*Taq PCR Master Mix, 0.5 mu L of primers and the balance of sterilizing double-distilled water; and the DNA template of the second-round PCR amplification system is prepared by diluting a first-round PCR amplification product for 10-20 times by volume. According to the invention, a first-round PCR amplification reaction system and a second-round PCR amplification reaction system are small, a dilution ratio of the first-round PCR product is small, a reagent required by the detection is also few, and the corresponding expense is low.

Description

technical field [0001] The invention relates to a PCR detection system for citrus huanglongbing asiatic species, in particular to a PCR detection system for citrus huanglongbing asiatic species nest and its application, and belongs to the field of molecular biology. Background technique [0002] At present, nested PCR (Nested-PCR) is a commonly used detection method for citrus huanglongbing, which has the advantages of higher sensitivity and stronger specificity than conventional PCR. The nested PCR reaction uses two sets of PCR primers to carry out two rounds of PCR amplification reactions. In the first round of amplification, the outer primers are used to generate amplification products, and the amplified products are subjected to the second round of amplification in the presence of embedded primers. increase. [0003] Although the nested PCR reaction increases the complexity of the amplification reaction, it reduces the possibility of amplifying multiple target sites and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12R1/01
CPCC12Q1/6848C12Q1/686C12Q1/70C12Q2531/113C12Q2549/119
Inventor 黄宏明廖惠红徐宁邢永秀方仁李鸿莉
Owner 广西壮族自治区农业科学院园艺研究所
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