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LAMP primer group and detection method for rapidly detecting corynespora cassiicola

A multi-primary corynebacterium and detection method technology, applied in the biological field, to achieve the effects of simple operation, shortening operation time, and overcoming technical difficulties

Inactive Publication Date: 2015-10-07
텐진인스티튜트오브플랜트프로텍션
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no report on the establishment of a LAMP detection method for Corynespora polymyces

Method used

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  • LAMP primer group and detection method for rapidly detecting corynespora cassiicola

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Embodiment 1

[0030] The LAMP detection of embodiment 1 Corynespora polymyxa

[0031] In order to verify the feasibility of the LAMP detection method, the pure culture of Corynespora multiprimum HB2 isolated from the specimen of Corynespora leaf spot of cucumber and identified by morphology and molecular biology was selected as the target strain, and its genomic DNA was used as the template . The total volume of the LAMP reaction system is 25 μL, including 2.5 μL 1×Thermol reaction buffer, 1.5 μL 1.4 mM dNTPs, 0.5 μL 0.2 μM forward outer primer CC-F3, 0.5 μL 0.2 μM reverse outer primer CC-B3, 2 μL 1.6 μM forward internal primer CC-FIP, 2 μL 1.6 μM reverse internal primer CC-BIP, 1 μL 0.8 mM betaine, 13 μL sterile double distilled water, 0.5 μL 8U / μL Bst DNA polymerase and 1.5 μL DNA template, to Add 1.5 μL of sterilized double distilled water as a negative control. The reaction conditions were constant temperature reaction in a water bath at 65°C for 60 minutes, and then 2 minutes at 80...

Embodiment 2

[0032]The LAMP primer-specific amplification test of embodiment 2 Corynespora polymata

[0033] A total of 19 strains of the target pathogen Corynebacterium multiprimum, non-target pathogens, and common pollutants were collected (see Table 1 for the test strains), and the genomic DNA of the 19 strains was used as the template DNA to be tested. The total volume of the LAMP reaction system is 25 μL, including 2.5 μL 1×Thermol reaction buffer, 1.5 μL 1.4 mM dNTPs, 0.5 μL 0.2 μM forward outer primer CC-F3, 0.5 μL 0.2 μM reverse outer primer CC-B3, 2 μL 1.6 μM forward internal primer CC-FIP, 2 μL 1.6 μM reverse internal primer CC-BIP, 1 μL 0.8 mM betaine, 13 μL sterile double distilled water, 0.5 μL 8U / μL Bst DNA polymerase and 1.5 μL DNA template, to Add 105 μL of sterilized double distilled water as a negative control. The reaction conditions were constant temperature reaction in a water bath at 65°C for 60 minutes, and then 2 minutes at 80°C to terminate the reaction. Test res...

Embodiment 3

[0038] The sensitivity test of the LAMP primer of embodiment 3 Corynespora polymycetes

[0039] In order to determine the sensitivity of the LAMP detection method of Corynespora polymata, the DNA concentration of the extracted pure culture of Corynespora polymata was measured with a spectrophotometer, adjusted to a DNA solution with a concentration of 1 μg / μl, and then carried out 10 times with DEPC water Gradual dilution, prepare standard DNA solutions with concentrations of 1μg / μl, 100ng / μl, 10ng / μl, 1ng / μl, 100pg / μl, 10pg / μl and 1pg / μl, take 1.5μl of each concentration standard solution as template DNA, Carry out LAMP reaction and result observation by the method of embodiment 1. The results showed that the established LAMP method could detect the DNA of Corynespora polymata at a concentration of 0.1ng / μl.

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Abstract

The invention discloses an LAMP primer group and a detection method for rapidly detecting corynespora cassiicola. A set of specific detection primer group is designed and selected for specific toxin cassiicolin gene sequence of cucumber corynespora cassiicola. The primer group consists of four specific primers, i.e. CC-FIP, CC-BIP, CC-F3 and CC-B3. The invention relates to a loop-mediated isothermal amplification (LAMP) detection method containing the primer group. The detection result can be directly observed by virtue of eyes or the color variation of SYBR GreenI is used as a judging standard to determine whether the corynespora cassiicola exists in a to-be-detected sample. The selected primer is high in sensitivity and specificity. The established detection method has advantages of good accuracy, short detection time and simple instrument and device; the process from the sample treatment to the report result can be completed only in 1.5h, a PCR instrument and an electrophoresis apparatus are not needed, the operation is simple, and compared with other PCR methods, the specificity is higher.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a detection primer set and a detection method for rapidly detecting Corynespora polyprime. Background technique [0002] Corynespora polymata ( Corynespora cassiicola (Berk&M.A.Curtis) C.T.Wei) is an important plant pathogenic fungus that can parasitize vegetables, trees and horticultural ornamental crops in tropical and subtropical regions. It mainly harms the leaves of plants, forming typical scabs, and can also infect roots, stems, flowers and fruits, causing leaf and fruit drop in severe cases. In recent years, the damage of Corynesporia leaf spot on vegetables in my country has increased year by year, mainly involving hosts such as cucumber, eggplant, tomato, kidney bean and other vegetables and economic crops such as rubber. The annual occurrence area exceeds 10 million mu and the loss exceeds 5 billion. It has become the main host An important limiting factor ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6844C12Q1/6895C12Q2531/119
Inventor 高苇王勇张春祥王万立郝永娟刘春艳霍建飞刘晓琳
Owner 텐진인스티튜트오브플랜트프로텍션
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