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Chinese chestnut blight nest-type PCR (polymerase chain reaction) detection kit and use method thereof

A technology of chestnut blight and detection kit, which is applied in the field of microorganisms and can solve the problem that molecular detection of C. parasiticaa has not yet been reported.

Inactive Publication Date: 2014-08-06
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the molecular detection of C.parasiticaa in China

Method used

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  • Chinese chestnut blight nest-type PCR (polymerase chain reaction) detection kit and use method thereof
  • Chinese chestnut blight nest-type PCR (polymerase chain reaction) detection kit and use method thereof
  • Chinese chestnut blight nest-type PCR (polymerase chain reaction) detection kit and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] The pathogenic bacteria and other tested strains were isolated from the branch tissue of the diseased Chinese chestnut. See Table 1 for the numbers, strains and sources.

[0022] Table 1 Tested strains and their sources

[0023]

[0024]

[0025] Note: "+" means there is a band after PCR amplification with primers ITS1 / ITS4 or ITS1 / ITS4 and ITSP1 / ITSP2, "-" means no band after PCR amplification with primers ITS1 / ITS4 or ITS1 / ITS4 and ITSP1 / ITSP2.

[0026] 1. Mycelia collection and DNA extraction of test bacteria

[0027] (1) Mycelia collection of test bacteria

[0028] After all the tested strains were purified and cultivated on PDA medium, fresh mycelium pieces were picked and inserted into PDB liquid medium. After 7 days of shaking culture at 25°C, they were filtered with sterilized gauze, rinsed with sterilized distilled water for 3 times, and the bacteria were collected. Shredded, freeze-dried and crushed into powder, stored at -20°C for later use.

[0029]...

Embodiment 2

[0046] Embodiment 2 Sensitivity detection

[0047] Dilute the genomic DNA concentration of C. parasitica to 30ng·μL with a 10-fold concentration gradient -1 , 3ng·μL -1 , 300pg·μL -1 , 30pg·μL -1 , 3pg·μL -1 , 300fg·μL -1 , 30fg·μL -1 , 3fg·μL -1 , 300ag·μL -1 , respectively for conventional PCR and nested PCR amplification detection.

[0048] 1. Conventional PCR amplification detection

[0049] The total volume of the mixed liquid of the PCR reaction system and program reaction is 25 μL, of which 2.5 μL 10×PCRBuffer (100 mmol·L -1 Tris-HCl, PH8.3, 500mmol·L -1 KCl), 10μmol L -1 Primers ITSP1 / ITSP2 each 0.8μL, 2μL 2.5mmol L -1 dNTP, 2μL 25mmol·L -1 MgCL2, 0.2 μL 5U·μL -1 Taq enzyme, template DNA extraction solution 1 μL, add sterilized double distilled water to make up 25 μL. A negative control was set up with sterilized water instead of template DNA. The reaction mixture was placed in a Mastercycler Personal PCR instrument (Eppendorf). The amplification progra...

Embodiment 3

[0054] Example 3 Detection of C.parasitica in chestnut stem tissue

[0055] Healthy tissues, non-infected tissues, newly infected tissues, typical diseased tissues, newly dead tissues and diseased plant residues were collected respectively, and genomic DNA was extracted to detect C. parasitica by conventional PCR and nested PCR respectively. The result is as Figure 4 , the results showed that both common PCR and Nested PCR could detect C. parasitica in typical diseased tissues and dead tissues, and Nested PCR could also detect pathogenic bacteria in primary disease, diseased plant remnants and bacteria-carrying tissues, It shows that Nest PCR has high sensitivity and specificity, and can be used for the rapid detection of chestnut blight in the field. In the later stage, the sequence comparison of PCR products and the isolation and identification of the pathogen also proved that C. parasitica was contained in the diseased tissue.

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Abstract

The invention discloses a Chinese chestnut blight nest-type PCR (polymerase chain reaction) detection kit and a use method thereof. The detection kit provided by the invention comprises (1) 10*PCR Buffer, wherein the components of the 10*PCR Buffer comprises 100 mmol / L<-1> of Tris-HCl at PH 8.3 and 500 mmol / L<-1> of KCl; (2) 2.5 mmol / L<-1> of dNTP; (3) 25 mmol / L<-1 > of MgCl2; (4) 5U / Mu L<-1> of Taq enzyme; (5) 10 Mu mol / L<-1> of primer ITS1 / ITS4, wherein the sequence of the ITS1 is 5'-TCCGTAGGTGAACCTGCGG-3' and the sequence of the ITS4 is 5'-TCCTCCGCTTATTGATATGC-3'; (6) 10 Mu mol / L<-1 > of premier ITSP1 / ITSP2, wherein the sequence of the ITSP1 is 5'-CCAGATACCCTTTGTGAACT-3' and the sequence of the ITSP2 is 5'-TCAGAGTCTTAGCGAGCC-3'; (7) sterilized double distilled water. According to the invention, the ITS1 / ITS4 and the ITSP1 / ITSP2 are adopted to form the nest-type PCR, 3 f g / Mu L<-1> genome DNA can be detected and the sensibility is increased by 1000 times. According to the adoption of the detection kit provided by the invention, pathogenic bacterium can be accurately detected in Chinese chestnut naturally pathogenetic tissues with the bacterium.

Description

technical field [0001] The invention relates to the field of microorganisms, in particular to a nested PCR detection kit for Phytophthora chestnut and a method for using the same. Background technique [0002] Chestnut blight (chestnut blight) is one of the world's famous forest diseases, and it is also one of the forestry quarantine pests in my country. The pathogen is C.parasitica, which mainly harms Chinese chestnut (Castanea mollissima), European chestnut (C.sativa Mill.), cone chestnut (C.henryi(skan) Rehd.et Wils.), American chestnut (Gastanea dentate Marsh .) and other beech plants. The pathogen mainly invades through wounds, and the main symptoms are rotten skin and ulcers on the main trunk, branches and twigs. In severe cases, the whole branch or even the whole plant dies. The disease was first discovered in the Americas in 1904. Due to its serious damage and rapid spread, it caused devastating disasters to European and American chestnut trees in the next half cen...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
CPCC12Q1/6848C12Q2531/113C12Q2549/119
Inventor 朱天辉麻文建朱涵明月李姝江谯天敏张静彭艳罗蓉
Owner SICHUAN AGRI UNIV
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