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PCR detection primers for pantoea ananatis as pathogeny of bacterial wilt disease of mulberries and application

A technology for detecting Pantoea pineapple and detection primers, which is applied in the determination/inspection of microorganisms, microorganisms, recombinant DNA technology, etc. It can solve the problems of weak detection technology, affecting disease quarantine and prevention and control, etc., and achieve good detection effect, high sensitivity, good specific effect

Active Publication Date: 2019-08-30
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, only molecular detection methods have been established for E.mori and Enterobacter cloacae, and the detection technology is relatively weak, which seriously affects the quarantine and prevention of diseases

Method used

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  • PCR detection primers for pantoea ananatis as pathogeny of bacterial wilt disease of mulberries and application
  • PCR detection primers for pantoea ananatis as pathogeny of bacterial wilt disease of mulberries and application
  • PCR detection primers for pantoea ananatis as pathogeny of bacterial wilt disease of mulberries and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Establishment of PCR Molecular Detection Method for Mulberry Bacterial Fusarium Wilt Pathogen Pantoea pineapple

[0065] 1. Experimental method

[0066] 1. Extraction of Pantoea pineapple genomic DNA

[0067] The DNA of bacterial pathogenic bacteria was extracted using the method of Ezup Column Bacterial Genomic DNA Extraction Kit. For the extraction method, please refer to the kit instruction manual.

[0068] 2. Primer design and reaction system and procedures

[0069] According to the BLASTn comparison between the Pantoea pineapple sequence and other Pantoea sequences published on NCBI, specific sequences were selected to design specific primers. The results of setting specific primers for the specific gene sequence of P. ananatis are shown in Table 5.

[0070] Table 5 Primer Results

[0071]

[0072] 3. Primer reaction temperature optimization

[0073] The PCR reaction amplification system is shown in Table 3. The amplification program was: pre-den...

Embodiment 2

[0079] Embodiment 2 A kind of mulberry bacterial wilt pathogen Pantoea pineapple specific detection kit

[0080] 1. Composition

[0081] Nucleotide sequence as shown in SEQ ID NO: 1-2 primers, 2×Taq Master Mix and ddH 2 O.

[0082] 2. How to use

[0083] 1. Extract the DNA of the sample to be tested

[0084] (1) Sample plant DNA extraction

[0085] The root tissue of the freshly collected mulberry plant was rinsed with sterile water, dried naturally, and cut into root segments of about 3-4 cm. Add 75% alcohol to the funnel on the clean workbench, soak for about 15 seconds, drain the alcohol, treat it with mercuric chloride for 5 minutes in the same way, and rinse it with sterile water for 3 to 4 times, take out the root segment and place it on the workbench to air dry , use sterile tweezers to tear off the cortex of the root segment to expose the wood, then use a sterile scalpel to scrape off the longitudinal strips of the xylem, put them in a sterile mortar, add about 5m...

Embodiment 3

[0093] Embodiment 3 Mulberry bacterial wilt pathogen PCR specific detection

[0094] 1. Experimental method

[0095]Enterobacter sp., Pseudomonas sp., Ralstonia solanacearum, Achromobacter sp., Kosakonia sp., White bacteria ( Ochrobactrum sp.), Acinetobacter sp., Lysobacter sp., Serratia marcescens, Sphingobacterium sp., Stenotrophomonas sp.), cupriavidus sp., Bacillus sp., Microbacterium sp., Arthrobacter sp., Burkholderia sp., Pantoea pineapple (P.ananatis) 18 bacterial strain genomes were used as templates, wherein the pantoea pineapple (P.ananatis) genome was used as a positive control, and sterile water was used as a template for a negative control. Detected according to the method of Example 2.

[0096] The DNA of bacterial pathogenic bacteria was extracted using the method of Ezup Column Bacterial Genomic DNA Extraction Kit. For the extraction method, please refer to the kit instruction manual.

[0097] 2. Experimental results

[0098] Depend on figure 2 It can be...

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Abstract

The invention discloses PCR detection primers for pantoea ananatis (pantoea ananatis) as pathogeny of bacterial wilt disease of mulberries and an application. The nucleotide sequence of the PCR detection primers for pantoea ananatis is as shown in SEQID NO:1-2. The invention provides specific PCR primers for detecting the pantoea ananatis as the pathogenic bacterium of the bacterial wilt disease of the mulberries, and through the primers, a detection method for the pantoea ananatis as the pathogenic bacterium of the bacterial wilt disease of the mulberries is established. The obtained primershave the advantages of being good in specificity and high in sensitivity, have excellent detection effects on DNA extracted from diseased plants and rhizosphere soil samples, which are infected with the pantoea ananatis as the pathogenic bacterium of the bacterial wilt disease of the mulberries, and have important significance on quick detection of the pantoea ananatis as the pathogenic bacteriumof the bacterial wilt disease of the mulberries in diseases of mulberries in a mulberry field.

Description

technical field [0001] The invention relates to the technical field of plant disease detection, and more specifically relates to a set of PCR detection primers and applications of Pantoea pineapple, the pathogen of mulberry bacterial wilt. Background technique [0002] Mulberry has a fundamental position and plays a leading role in the development and transformation of the sericulture industry. With the transformation of the mulberry industry, the basic research of mulberry will become an important support for the development and innovation of the mulberry industry under the background of the comprehensive utilization of mulberry resources, the continuous development of the mulberry industry, the completion of mulberry genome sequencing, and the comprehensive development of functional gene research. [0003] As one of the most important mulberry bacterial diseases, mulberry bacterial wilt disease is common and serious in silkworm regions in Zhejiang and South China. The dis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/11C12R1/01
CPCC12Q1/689
Inventor 刘吉平王继承
Owner SOUTH CHINA AGRI UNIV
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