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Molecular detection primer for sweet potato black rot germs and application of molecular detection primer

A black rot fungus and molecular detection technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve long-term problems and achieve strong specificity, reliable results, and high sensitivity Effect

Active Publication Date: 2013-04-17
漳浦县六鳌全域有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to be able to correctly grasp the occurrence of the disease and take corresponding measures for prevention and control in a timely manner, it is necessary to quickly determine whether the sample with symptoms in the field is caused by an epidemic pathogen. However, the traditional identification method of pathogenic bacteria takes a long time. In order to apply to production To provide a more rapid and reliable detection method, the present invention is based on the gyrB Gene sequence design primers to establish a PCR detection method for sweet potato black rot fungus

Method used

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  • Molecular detection primer for sweet potato black rot germs and application of molecular detection primer
  • Molecular detection primer for sweet potato black rot germs and application of molecular detection primer
  • Molecular detection primer for sweet potato black rot germs and application of molecular detection primer

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Effect test

Embodiment 1

[0035] Embodiment 1: Primer is specific to the amplification of sweet potato black rot bacterium

[0036] 1. Specific Detection of Sweet Potato Black Rot Pathogen

[0037] The DNA extraction method of each strain was carried out according to the above-mentioned SDS-CTAB method, and the PCR reaction system was 25 μL, including 2.5 μL 10×PCR reaction buffer, 2.5 mmol / L Mg 2+ , 150 μmol / L dNTPs, 1.25U Taq DNA polymerase, primers DyB1F and DyB2R each 0.2 μmol / L and 10 ng template DNA, the rest is ddH 2 O; PCR reaction conditions were: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 35 s, annealing at 56°C for 35 s, extension at 72°C for 40 s, 30 cycles, and extension at 72°C for 5 min.

[0038] 2. Test results

[0039] Specificity detection results: In addition to the 509 bp product that can be specifically amplified by sweet potato black rot, the detected potato black rot, rice base rot, banana bacterial soft rot, radish soft rot, sweet potato The correspondi...

Embodiment 2

[0040] Embodiment 2: Sensitivity detection of primers to DNA of sweet potato black rot bacteria

[0041] 1. DNA extraction and dilution of detection bacteria: The DNA extraction method was carried out according to the above-mentioned SDS-CTAB method, and the DNA solution of sweet potato black rot bacteria was serially diluted to 100 ng / μL, 10 ng / μL, 1 ng / μL, 100 pg / μL, 10 pg / μL, 1 pg / μL, 100 fg / μL, 10 fg / μL, and then pipette 1 μL for PCR detection.

[0042] 2. Sensitive detection of DNA of sweet potato black rot fungus

[0043] PCR reaction system 25 μL, including 2.5 μL 10×PCR reaction buffer, 2.5 mmol / L Mg 2+ , 150 μmol / L dNTPs, 1.25U Taq DNA polymerase, primers DyB1F and DyB2R each 0.2 μmol / L and template DNA 1 μL, the rest is ddH 2 O; PCR reaction conditions were: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 35 s, annealing at 56°C for 35 s, extension at 72°C for 40 s, 30 cycles, and extension at 72°C for 5 min. Amplified products were detected by e...

Embodiment 3

[0045] Embodiment 3: the detection of sweet potato black rot pathogen in diseased plant tissue

[0046] 1. Sample collection: The plant tissue samples of diseased sweet potato were collected from the planting base in Xindian, Fuzhou.

[0047] 2. DNA extraction and detection

[0048]The DNA of the diseased plant tissue was obtained by the DNA rapid extraction method of the sweet potato black rot pathogen in the diseased tissue. The specific method was to take 300 mg stem tissue, cut it into pieces with medical surgical scissors, and add 1 mL of 0.1 mol / L and pH 8. 0 Tris-HCl buffer solution, let stand for 15 min, centrifuge at 3000 rpm for 2 min, remove the supernatant to another new 1.5 mL Eppendorf tube, centrifuge at 12000 rpm for 2 min, discard the supernatant, add 50 μL of 0.1 mol / L. Tris-HCl with a pH of 8.0, buffer solution, placed on a vortex shaker to shake the precipitate into a suspension, boiled in boiling water for 8 minutes, centrifuged at 12000 rpm for 5 mi...

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Abstract

The invention provides a molecular detection primer for sweet potato black rot germs and an application of the molecular detection primer. The specific primer comprises an upstream primer DyB1F: 5'-TTCATCGTCAGGAATACCG-3' and a downstream primer DyB2R: 5'-GACCTTGGCTTTCTTGCTGTA-3';; and a specific amplification product with 509 bp of segment length can be amplified from pure DNA (deoxyribonucleic acid) of the sweet potato black rot germs, a tissue of a germ-carrying disease plant and soil by PCR (Polymerase Chain Reaction) amplification and agarose gel electrophoresis so as to detect the sweet potato black rot germs quickly. The specific molecular detection primer and the application thereof can be used for the quick, sensitive and specific detection of the sweet potato black rot germs in the germ-carrying soil, sick bodies, the disease plants and seedlings in the production practice, can also be used for the early diagnosis of field diseases and the monitoring and identification of the germs, and provides the control of the diseases caused by the sweet potato black rot germs with reliable technical and theoretical bases.

Description

technical field [0001] The invention belongs to the field of crop disease detection, identification and prevention technology, and more specifically relates to a molecular detection primer for sweet potato black rot fungus and its application. Background technique [0002] sweet potato (Ipomoea batatas (L.) Lam. ) is one of the world's food, energy and industrial raw material crops, and my country is the largest sweet potato planting country in the world. Sweet potato black rot is caused by Dickeya dadantii A disease caused by infection can seriously damage the stems and roots of sweet potatoes, causing a large number of dead seedlings and seriously affecting the production of sweet potatoes. Fang Shumin reported the occurrence of sweet potato bacterial black rot in Putian, Hui'an and Jinjiang, Fujian Province in 1991, and the pathogen was identified as Erwinia sp., the serious loss amounted to more than 80%. In 2003, Luo Kechang conducted a survey on the harm and pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 邱思鑫刘中华邱永祥徐永清余华
Owner 漳浦县六鳌全域有限公司
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