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Primer pair for detecting sweet potato stem rot pathogen as well as detection method thereof

A primer pair, sweet potato technology, applied in the biological field, can solve the problems of inapplicability of specific detection, difficulty in distinguishing different species of Dickeya, and difficulty in detection of sweet potato stem rot fungus, etc.

Active Publication Date: 2018-09-28
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is difficult to distinguish different species of Dickeya by traditional morphological and physiological and biochemical analysis methods, and the previous molecular detection methods for E. chrysanthemi are not suitable for specific detection of D. dadantii
And because the interspecific differences of Dickeya genus are small, it is difficult to design species-specific primers for Dickeya dadantii. At present, there are no relevant reports in the literature at home and abroad. Therefore, it is difficult to apply PCR methods to sweet potato. stem rot detection

Method used

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  • Primer pair for detecting sweet potato stem rot pathogen as well as detection method thereof
  • Primer pair for detecting sweet potato stem rot pathogen as well as detection method thereof
  • Primer pair for detecting sweet potato stem rot pathogen as well as detection method thereof

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Experimental program
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Effect test

Embodiment 1

[0035] Specific gene sequence screening and primer design

[0036] 1) Specific gene screening

[0037]According to the genome sequence of P. spp. in the NCBI genome database, its specific genes were found out by pan-genome analysis, and the specificity of the found genes was verified by local Blast. The specific gene sequence is shown in SEQ ID No.1.

[0038] 2) Primer design

[0039] For the specific gene sequence, the primers are designed by Primer-BLAST, an online design specific oligonucleotide primer tool. The upstream primer is shown in SEQ ID No.2, and the downstream primer is shown in SEQ ID No.3. The specific company that produces the primers is Hangzhou Qingke Biotechnology Co., Ltd.

Embodiment 2

[0041] Establishment of Molecular Identification Methods

[0042] 1) DNA extraction of the sample to be tested

[0043] Pick a single colony of the strain to be tested in 5mL NA liquid medium, 180r min -1 , shake culture at 30℃ for 12h, until the concentration is 10 8 CFU mL -1 , centrifuged to collect the bacterial cells, using the TIANGEN Bacterial Genomic DNA Extraction Kit to extract the bacterial genomic DNA, and UV spectrophotometer Nanodrop 2000 to detect the quality and concentration of the extracted DNA. Standby at -20°C.

[0044] 2) Establishment of PCR amplification system and method

[0045] The PCR amplification reaction system is a 25 μL system, specifically: 1 μL DNA template, 1 μL upstream primer: Dda-F, 1 μL downstream primer: Dda-R, 12.5 μL Taq PCR Mix, 9.5 μL ddH 2 O.

[0046] The PCR amplification program was: pre-denaturation at 94°C for 3 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 10 s, and 35 cycles; exten...

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Abstract

The invention discloses a primer pair for detecting sweet potato stem rot pathogen as well as a detection method thereof, and belongs to the field of a biological technology. The primer pair comprisesan upstream primer and a downstream primer; the nucleotide sequence of the upstream primer is as shown in SEQ ID No.2; and the nucleotide sequence of the downstream primer is as shown in SEQ ID No.3.A specific gene fragment of dardenticella is obtained through bioinformatics means, a specific primer pair for performing PCR amplification on the gene fragment is designed, accurate authentication of the dardenticella is realized by the primer pair, and the lowest detection concentration of the sweet potato stem rot pathogen DNA reaches to 0.5 ng / uL. By the detection method provided by the invention, the sweet potato stem rot pathogen in a sweet potato sample can be authenticated rapidly and accurately, and complex operation such as pathogen culture and character analysis is not needed.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a pair of primers for detecting sweet potato stem rot and a detection method thereof. Background technique [0002] Sweet potato is the sixth largest staple food in the world, and China is the largest producer of sweet potato in the world. From 2006 to 2010, the planting area of ​​sweet potato remained stable at 3.70×106hm 2 About 45.1% of the world's sweet potato planting area, the average annual output is about 7.8×107t, accounting for about 75% of the world's sweet potato production, the total output is second only to rice, wheat and corn in China, and occupies an important position in China's national economy. [0003] Sweet potato bacterial stem rot was first discovered in the United States in 1974, and later also reported in Japan and Venezuela in South America. In recent years, bacterial stem rot has successively occurred in sweet potatoes in Fujian, Guangdong, Jiangxi, Guang...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 吴秀芹洪纤纤杨敏姚榕王艳丽孙国昌安千里李斌
Owner ZHEJIANG UNIV
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