Cryphonectria parastitica (Murr.) barr molecular detection kit and use method thereof

A chestnut Phytophthora bacterium and molecular detection technology, applied in the field of microbiology, can solve the problems of difficulty in accurate identification of similarity, inability to achieve rapid and accurate detection and identification of pathogenic bacteria, false positives, etc., to achieve rapid and accurate early diagnosis and overcome poor stability , the effect of improving accuracy

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

However, the acquisition of its sequence still requires a series of cumbersome steps such as isolation, purification and culture, DNA extraction, PCR amplification, recovery and cloning, and sequencing of the strain, which still cannot achieve rapid and accurate detection and identification of pathogenic bacteria.
Some fungal rDNA-ITS sequence similarities are relatively high, and it is difficult to accurately identify them only by designing primers based on existing ITS sequences
Therefore, the detection of some fungi only relies on rDNA-ITS sequence analysis technology to design primers, which may produce false positives

Method used

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  • Cryphonectria parastitica (Murr.) barr molecular detection kit and use method thereof
  • Cryphonectria parastitica (Murr.) barr molecular detection kit and use method thereof
  • Cryphonectria parastitica (Murr.) barr molecular detection kit and use method thereof

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Experimental program
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Embodiment

[0027] Main reagents used: DNA extraction kit and recovery kit were purchased from Tiangen Biochemical Technology Company, DH5αE.coli Competent and Premix were purchased from TaKaRa Company, primer synthesis and PCR product sequencing were completed by Shanghai Sangon Bioengineering Company.

[0028] figure 1 It is the implementation diagram of the dual PCR detection technology for Phytophthora chestnut.

[0029] 1. Design of double PCR primers for rapid molecular detection of Phytophthora chestnut

[0030](1) Primers in the rDNA-ITS region: Purify and cultivate the Chestnut Phytophthora bacterium isolated from chestnut cultivation areas in Chongqing, Sichuan, Ya'an, Sichuan, and Luzhou, Sichuan. First, inoculate the tested strain on PDA medium, and place it in a dark condition in an incubator at 25°C Cultivate under low temperature for 3 days, then pick 3~4 pieces of bacteria cakes about 5mm from the edge of the colony, inoculate them in a conical flask containing 100ml of p...

Embodiment 2

[0091] Example 2 Detecting Field Diseased Plants with Chestnut Phytophthora Molecular Detection Kit

[0092] 1. Genomic DNA extraction:

[0093] Genomic DNA was extracted by the CTAB method as follows:

[0094] 1) Cut out a section of 50-100 mg suspected chestnut blight disease tissue with a scalpel, disinfect the surface with 70% alcohol, rinse it under distilled water, dry the water with absorbent paper, transfer it to a mortar and grind it quickly with liquid nitrogen (for easy Grinding can be properly cut into powder, put into a 1.5mL centrifuge tube;

[0095] 2) Add 900 μL of CTAB solution and 90 μL of 10% SDS after preheating at 60°C, and mix well;

[0096] 3) 60°C water bath for 1 hour, in the middle every 10mm upside down and mix once;

[0097] 4) Centrifuge at 12000rpm for 10min;

[0098] 5) Add 700 μL volume of phenol:chloroform:isoamyl alcohol (25:24:1) to the supernatant, mix well, and let stand for 10 minutes;

[0099] 6) Centrifuge at 12000rpm for 10min;

...

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Abstract

The invention discloses a Cryphonectria parastitica (Murr.) barr molecular detection kit and a use method thereof. The detection kit provided by the invention comprises 10 Mu mol/L of a primer CP1/CP2, and 10 Mu mol/L of a primer SC1/SC2 , dd H2O and Premix, wherein each of CP1/CP2 is 1 Mu L; each of SC1/SC2 is 1 Mu L, dd H2O is 7.5 Mu L and Premix is 12.5 Mu L; the sequence of CP1 is 5'-CGGCCCACTAAACTCTTTGT-3'; the sequence of the CP2 is 5'-ATTTCAGGGCCTACCGTTTT-3', the sequence of the SC1 is 5'-AACGGTGACCCTTCTGGGGC-3'; the sequence of the SC2 is 5'-AACGGTGACCGCAAAGGACTC-3'. The Cryphonectria parastitica (Murr.) barr molecular detection kit overcomes the defects that the RAPD (Random amplification polymorphism DNA) is poor in stability and repeatability and the like, and the disease detection accuracy is improved simultaneously. The Cryphonectria parastitica (Murr.) barr molecular detection kit can quickly and accurately identify and diagnose the Cryphonectria parastitica (Murr.) barr at an early stage and has significance for controlling Cryphonectria parastitica (Murr.) barr detection and prevention and control of disease.

Description

technical field [0001] The invention relates to the field of microbes, in particular to a molecular detection kit for Phytophthora chestnut and its application method. Background technique [0002] Chestnut blight is one of the world's famous forest diseases, and it is a devastating disease of chestnut trees, which brings huge losses to production. Chestnut blight threatened the American chestnut species with total extinction in the nineteenth century, and is the only typical example of a plant disease destroying a natural species. In 1913, American scholar Shear C.L first discovered chestnut blight in eastern China. Since then, blight has occurred in Hebei, Shandong, Henan, Jiangsu, Zhejiang, Shaanxi, Hunan, Guangdong, Jiangxi and other places. The disease is caused by Cryphonectria parasitica (Murr.) barr. Chestnut blight mostly occurs near the surface of the trunk and at the base of main branches. Young trees have a high incidence and death rate in the same year, and on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/686C12Q2537/143C12Q2535/139
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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