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Primer group for detecting field pectobacterium carotovorum subsp and kit and method for extracting and detecting DNA of pectobacterium carotovorum subsp

A technology of pectin bacillus and soft rot pectin, which is applied in the field of gene detection and diagnosis and nucleic acid extraction, can solve the problems of lack of rapid extraction of nucleic acid of soil-borne pathogenic bacteria, and the assumption and verification of integrated detection methods, so as to avoid false positive problems, Simple operation and easy operation effect

Active Publication Date: 2019-09-10
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0021] At present, there is still a lack of conception and verification of an integrated method for the rapid extraction and detection of nucleic acid of soil-borne pathogens that can be applied in the field

Method used

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  • Primer group for detecting field pectobacterium carotovorum subsp and kit and method for extracting and detecting DNA of pectobacterium carotovorum subsp
  • Primer group for detecting field pectobacterium carotovorum subsp and kit and method for extracting and detecting DNA of pectobacterium carotovorum subsp
  • Primer group for detecting field pectobacterium carotovorum subsp and kit and method for extracting and detecting DNA of pectobacterium carotovorum subsp

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Experimental program
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Effect test

Embodiment 1

[0112] Example 1 is used to detect the primer set of field soft rot pathogen

[0113] Based on the single base difference phenomenon of pathogen-specific targets, combined the single base difference phenomenon of target pathogen-specific targets with loop-mediated isothermal amplification technology (LAMP), with the help of NCBI online database, DNAMAN was used to conduct soft rot pathogen stewardship Gene, ITS spacer, pmrA gene and other characteristic gene sequences were analyzed and compared to determine the differential sites that can be stably inherited, and a specific detection method for Pectobacterium carotovorum subsp. carotovorum was designed and screened. Optimal Primer Combination. The primer composition capable of specifically detecting Pectobacterium carotovorum subsp. carotovorum consists of the following:

[0114] Forward outer primer Pcc-F3: 5'-GGCCAAAGAAGCAGACGC-3',

[0115] Reverse outer primer Pcc-B3: 5'-GGCTTGGCAAGGTAATCGT-3';

[0116] Forward internal ...

Embodiment 2

[0121] Based on the above-mentioned experimental screening primer set, the detection kit for field Pectinobacillus soft rot subsp. solution, eluent; the detection box includes the primer set and detection reaction solution according to claim 1; wherein, the primer set of Pectobacterium carotovorum subsp.carotovorum is

[0122] Forward outer primer Pcc-F3: 5'-GGCCAAAGAAGCAGACGC-3',

[0123] Reverse outer primer Pcc-B3: 5'-GGCTTGGCAAGGTAATCGT-3';

[0124] Forward internal primer Pcc-FIP:

[0125] 5'-GCCCATCTTCATCCGGTAAGCCCATCAGCAGCGCGCATTA-3',

[0126] Reverse internal primer Pcc-BIP:

[0127] 5'-ATCATTACCAGCATCCCGTGCTCGCTGACGCGATCGTTTA-3'.

[0128] As a preferred solution, the detection kit consists of a primer set and a detection reaction solution; the detection reaction solution includes: 10x isothermal amplification reaction buffer, 100mM MgSO4, 10mM dNTP Mix, 5M betaine, 8U / μL Bst 2.0 DNA polymerase , Hydroxynaphthol Blue (HNB).

[0129] The preparation method of each m...

Embodiment 3

[0164] Embodiment 3 above-mentioned detection kit is directly detected to plant tissue

[0165] Tissue samples (16) of Chinese cabbage with different degrees of disease were collected in the field, and the above-mentioned detection kits were used for rapid detection:

[0166] POCT detection and routine PCR detection were performed on the tissue samples of Chinese cabbage with obvious disease (8) and Chinese cabbage without obvious symptoms (8).

[0167] Such as Figure 7 Shown: All the obviously susceptible Chinese cabbage tissue samples were positive, and 25% of the Chinese cabbage tissue samples without obvious symptoms were positive; conventional PCR results showed that 62.5% of the obviously susceptible Chinese cabbage tissue samples were positive, and no Tissue samples of Chinese cabbage with obvious symptoms were all negative.

[0168] It can be seen from this that: (1) the above-mentioned kit can directly detect not only pathogenic bacteria, but also plant tissue samp...

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Abstract

The invention discloses a primer group for detecting field pectobacterium carotovorum subsp and a kit and method for extracting and detecting DNA of the pectobacterium carotovorum subsp. The kit combines a sample nucleic acid rapid extracting technology and a loop-mediated isothermal amplification technology to establish a bacterial soft rot pathogenic bacterium product based on a soft rot pathogenic bacterium specific target single-base difference phenomenon. The detection method has the advantages that soft rot pathogenic bacteria, plant tissue and soil samples can be rapidly detected separately, nucleic acid extraction can be completed within 30 seconds, detection only needs 30-40 minutes, detection is rapid and accurate, no expensive instrument is needed, the cost is low, and the detection method can be directly used for real-time and on-site detection in the field, which facilitates dynamic monitoring of the soft rot pathogenic bacteria in soil, facilitates the development of popular prediction and early warning work of the soft rot, and facilitates early disease prevention and treatment.

Description

technical field [0001] The invention relates to the fields of nucleic acid extraction and gene detection and diagnosis, in particular to a primer set for detecting field Pectinobacillus soft rot subspecies carotene, a rapid detection kit and a method thereof. Background technique [0002] At this stage, with the development of protected areas, continuous cultivation of high value-added crops, straw returning to the field, and promotion of no-tillage techniques, a large number of pathogenic bacteria accumulate in the soil, causing soil-borne diseases to occur. Soil-borne diseases have the characteristics of various types, wide distribution, rapid spread, great harm, and difficult control. Pathogens can overwinter in the soil and continue to attack crops in the coming year, and this cycle makes the disease more serious. In general, soil-borne diseases damage the roots and stems of plants. In the early stage of crop growth, the roots and stems of seedlings can rot and die; Us...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12Q1/6844C12Q1/6806C12N15/11C12R1/01
CPCC12Q1/6806C12Q1/6844C12Q1/689C12Q2531/119C12Q2547/107C12Q2527/125
Inventor 游红陈若琳李建洪万虎何顺朱福兴马洪菊
Owner HUAZHONG AGRI UNIV
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