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Nested PCR detection kit and application thereof to detection of verticillium dahliae

A detection kit and a technology for Fusarium wilt bacteria are applied in the fields of identification and prevention, and detection of garden plant diseases, and can solve the problems of difficult detection, low proportion of cotton Verticillium wilt bacteria, and unsuitability for rapid detection of Verticillium dahliae.

Inactive Publication Date: 2018-07-24
BEIJING INST OF LANDSCAPE ARCHITECTURE
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in actual production, due to the complexity of the physical and chemical properties of the soil and the ecosystem, the proportion of Verticillium dahliae in the soil is very low, and it is difficult to detect its existence by conventional PCR methods.
In addition, due to the different plant genome sequences and the rapid mutation of pathogenic bacteria, the existing detection technology is not suitable for the rapid detection of Verticillium dahliae

Method used

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  • Nested PCR detection kit and application thereof to detection of verticillium dahliae
  • Nested PCR detection kit and application thereof to detection of verticillium dahliae
  • Nested PCR detection kit and application thereof to detection of verticillium dahliae

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1: Nested PCR detection of cotinus fusarium wilt in plant rhizosphere soil

[0040] Detection of Fusarium wilt fungus in soil. Soil samples include: (1) normal pure forest sumac rhizospheric soil; (2) diseased pure forest sumac rhizospheric soil; (3) the rhizospheric soil of S. arborvitae; (3) the rhizospheric soil of Spiraea trilobata; 4) soybean rhizosphere soil; (5) sandy cypress rhizosphere soil. Each soil sample was tested in triplicate. Specific steps are as follows:

[0041] Extraction of DNA from plant rhizosphere soil

[0042] About 0.25 g of the soil sample was taken, ground with liquid nitrogen, and DNA was extracted according to the operation steps of the Tiangen Soil Genomic DNA Extraction Kit (DP336) to obtain a DNA sample.

[0043] First round of PCR amplification

[0044] Use primer P1 and primer P2 (see the table below for the specific sequence) to amplify the DNA sample extracted in (1) (as template DNA). The PCR reaction system is: to...

Embodiment 2

[0053] Example 2: Nested PCR detection of cotinus fusarium wilt in plant leaves

[0054] Extraction of plant leaf DNA

[0055] About 0.25 g of plant leaf samples were taken, ground with liquid nitrogen, and DNA was extracted according to the operation steps of the high-efficiency plant genomic DNA extraction kit (DP350) to obtain DNA samples.

[0056] First round of PCR amplification

[0057] Use primer P1 and primer P2 to carry out PCR amplification on the extracted DNA sample (as template DNA). The PCR reaction system is: total reaction volume 25 μL: template DNA 1 μL, 5U / μL Taq enzyme 0.2 μL, 10×PCR reaction buffer (containing 100 mM Tris-HCl buffer solution with pH 8.3, 15 mM MgCl2 and 500 mM KCl) 2.5 μL, 2.5 mmol / L d NTPs mixture 2.0 μL, 10 μmol / L primers P1 and P2 each 1 μL, sterile double distilled water Make up to 25 μL. The reaction mixture was amplified on a BIOMETRAT-Gradient gradient PCR instrument. The reaction program was: 94°C pre-denaturation for 5 minut...

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Abstract

The invention provides a nested PCR detection kit. The kit comprises a primer P1 and a primer P2 for first-round PCR amplification, and a primer PN1 and a primer PN2 for second-round PCR amplification, wherein the primers P1, P2, PN1 and PN2 have the same or similar amplification functions shown as SEQ ID NO. 1-4 respectively in PCR amplification reactions. The invention further provides the application of the kit to detection of the verticillium dahlia. The invention further provides a method for detecting the verticillium dahlia by using the kit. The kit can quickly detect the verticillium dahlia with high sensitivity and high accuracy.

Description

technical field [0001] The invention belongs to the technical field of garden plant disease detection, identification and prevention, and in particular relates to a detection kit for detecting cotinus fusarium wilt and a use method thereof. Background technique [0002] Cotinus coggygria (Cotinus coggygria), also known as Cotinus coggygria or red leaves, is a plant of the genus Cotinus coggygria in the Anacardaceae family; deciduous shrubs or small trees, with a plant height of 3 to 5 meters, and the leaves are bright red after frost in autumn. It is of great ornamental value and economic value. As the main red leaf tree species in Beijing Xiangshan Park, it attracts a large number of Chinese and foreign tourists and plays an irreplaceable role in the inheritance of red leaf culture. [0003] However, since the 1980s, due to the infection of Verticillium dahliae (Verticillium dahliae), large areas of Cotinus cotinus in Beijing, Shandong and other places have died. Thousands...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6848C12Q1/6895C12Q1/04
CPCC12Q1/6848C12Q1/6895C12Q2549/119
Inventor 周江鸿夏菲车少臣
Owner BEIJING INST OF LANDSCAPE ARCHITECTURE
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