Method and application of molecular rapid detection of mango malformation pathogen (fusarium mangiferae)

A technology for pathogenic bacteria and malformation, which is applied in the field of rapid molecular detection and application of mango malformation pathogen (Fusarium mangiferae), can solve the problems of simplicity, lack of convincing generality and specificity, and insufficient operation, so as to achieve simple operation and reliable results Guaranteed, reliable results

Inactive Publication Date: 2016-06-22
SOUTH SUBTROPICAL CROPS RES INST CHINESE ACAD OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

This method mainly compares the difference between nested PCR and ordinary PCR detection results. Although nested PCR has high sensitivity, its operation is not as simple as ordinary PCR, and the detection is not convincing in terms of universality and specificity.

Method used

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  • Method and application of molecular rapid detection of mango malformation pathogen (fusarium mangiferae)
  • Method and application of molecular rapid detection of mango malformation pathogen (fusarium mangiferae)
  • Method and application of molecular rapid detection of mango malformation pathogen (fusarium mangiferae)

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Experimental program
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Effect test

Embodiment 1

[0041] The acquisition of embodiment 1 bacterial strain specific primer

[0042] Using the genomic DNA of the target strain MG06 and plant pathogenic Fusarium (FM1-FM7) as a template, and the genomic DNA of healthy mango as a control, the primers were screened by ISSR molecular marker technology, and ISSR primers (BDB (CA) 7 ) has good reproducibility, and can stably amplify a specific band (479bp) at the channel of the target strain, and there is no band displayed in the channel of the mango genome ( image 3 ). The specific bands were gel-cut and recovered, and the sequencing was completed by Invitrogen. The sequence of the target fragment was compared with the NCBI database, and it was found that only Fusarium fujikuroi (HF679027) was similar to its sequence, with a homology of 87%. According to the whole genome sequence of Fusarium fujikuroi (HF679027) and the measured target strain sequence, multiple sets of primers were designed, among which primers W342 (ACAACTCCGCACC...

Embodiment 2

[0043] Specific detection of embodiment 2 primers W342 and W1772

[0044] According to primers W342 and W1772, determine the PCR reaction system and parameters: the total volume of the amplification reaction is 25 μL, 2.5 μL of 10×PCR buffer (TaKaRa), 2 μL of 10 mmol / L dNTPs, 0.5 μL of template (10ng / μL), mixed primers (20 μmol / L) 1 μL, Taq enzyme (5U / μL) (TaKaRa) 0.13 μL, add ddH 2 O to 25 μL of total system. PCR reaction conditions: 94°C for 5min; 94°C for 1min, 50°C for 30s, 72°C for 1min, 45 cycles; 72°C for 5min. After the reaction, 5 μL of the PCR product was electrophoresed on a 1.0% Agarose gel (100V, 35min), detected by UV, and photographed and recorded by the gel imaging system. Test results( Figure 4 ) shows that only the specific band appears in the channel of the target strain, but there is no band in other comparison strains and controls. At the same time, the specific band was sent to Invitrogen for sequencing after gel cutting, and a specific sequence with...

Embodiment 3

[0045] Sensitivity detection of embodiment 3 primers W342 and W1772

[0046]The sensitivity test is divided into two parts: the first part detects mango malformation disease MG6 genomic DNA. The detection template content is divided into 4 gradients: 10ng, 1ng, 0.1ng, 0.01ng; the second part detects the mixed template of healthy mango and deformed bacteria MG6 genomic DNA, so that the deformed bacteria MG6 genomic DNA content is: 10ng, 1ng, 0.1 ng, 0.01ng. Test results( Figure 5 ) shows that: in the two detection tests, the method can detect the lowest fungal DNA content is 10pg, and the sensitivity is high.

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Abstract

The present invention discloses a rapid molecule detection method for a mango plant disease Fusarium mangiferae, and an application thereof, and belongs to the technical field of crop plant disease detection, identification and prevention and control. According to the present invention, an ISSR molecular marker is mainly adopted to screen primers and a target fragment, the target fragment sequence is analyzed, a pair of SCAR special primers (W342:5'-ACAACTCCGCACCCTCAATG-3' and W1772:5'-GGACCCTCAGACTGCCCAAT-3') is designed to establish the rapid molecular detection method, a PCR reaction is performed on DNA contained in a sample with the method, and it is determined that the sample contains the mango malformation disease Fusarium mangiferae when the 1376bp specific PCR product is presented; the method has characteristics of simple operation and strong specificity, wherein the lowest fungal DNA detection content is 10 pg; and the detection method is suitable for high sensitivity and rapid detection of the mango malformation disease Fusarium mangiferae and the field mango tissue carrying Fusarium mangiferae, and provides the reliable theoretical basis and the technical method for early diagnosis and timely prevention of the mango malformation disease.

Description

technical field [0001] The present invention relates to a method and application of rapid molecular detection of mango malformation pathogen (Fusarium mangiferae), which uses a pair of specific primers and polymerase chain reaction (PCR) molecular biology technology to detect mango malformation pathogen (Fusarium mangiferae) with high sensitivity. The rapid detection can be used for the early diagnosis of mango deformity disease in the field and the detection and identification of germs, and belongs to the field of detection, identification and prevention of crop diseases. Background technique [0002] Mango malformation disease (mango malformation disease, MMD), also known as cluster disease, cluster bud disease, is caused by Fusarium or its complex. At present, there are 6 kinds of pathogenic bacteria that are widely reported at home and abroad and verified by Koch's law, namely: Fusarium subglutinans, Fusarium mangiferae, Fusarium proliferatum, Fusarium sterili hyphosum, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q2531/113
Inventor 吴婧波柳凤詹儒林李国平赵艳龙常金梅何衍彪
Owner SOUTH SUBTROPICAL CROPS RES INST CHINESE ACAD OF TROPICAL AGRI SCI
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