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Nested PCR detection method of oak wilt

A detection method, the technology of oak withering, applied in the biological field, can solve the problems that have not been reported, and achieve the effect of strong reliability, simple procedure, and shortened quarantine period

Inactive Publication Date: 2011-09-21
ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are no reports about the Nested PCR detection method of Fusarium wilt, or even other molecular detection methods at home and abroad.

Method used

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  • Nested PCR detection method of oak wilt
  • Nested PCR detection method of oak wilt
  • Nested PCR detection method of oak wilt

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] The nested (Nested) PCR detection of embodiment 1 oak wilt

[0033] Follow these steps:

[0034] 1. Sample DNA Extraction

[0035] 1.1 DNA extraction from mycelium samples

[0036] 1.1-1 Test strain culture and mycelium collection

[0037] The tested strains were inoculated on PDA medium, cultured in the dark at 20-25°C for 10 days, the mycelium was gently scraped off with an inoculation needle, placed in a centrifuge tube, freeze-dried, and stored at -20°C for future use.

[0038] 1.1-2 Use the CTAB method to extract mycelial DNA, the specific operation is as follows:

[0039] 1) Take 0.5g of frozen mycelium, put it in a 2.0ml flat-bottomed centrifuge tube, add 2 steel balls with a diameter of 3mm, close the cap, put it on the Oscillating Mill MM400 grinder, 30f / min, and grind for 3 minutes;

[0040]2) Take out the steel ball, add 500 μl CTAB, freeze in liquid nitrogen for 2 minutes, then bath in 75°C water bath for 2 minutes, repeat twice, and finally melt in 75°C...

Embodiment 2

[0070] Embodiment 2 Specific Amplification Sensitivity Test

[0071] Dilute the DNA of strain Ceratocystis fagacearum ATCC 200423 into different concentration gradients, 1×10 2 pg / μl, 10pg / μl, 1pg / μl, 1×10 -1 pg / μl, 1×10 -2 pg / μl, take 1μl of DNA of each concentration as a template, and use only specific primers CF01 / CF02 for PCR amplification once, and the electrophoresis results after amplification are shown in figure 2 . The same results were obtained by repeating the amplification three times.

[0072] figure 2 Among them, M is a 2000-bp DNA marker; 1-5 represent the amplification products with DNA concentrations of 100pg, 10pg, 1pg, 0.1pg, and 0.01pg in the 25μl reaction system; 6 is the negative control. figure 2 It was shown that the minimum detectable bacterial DNA concentration was 10 pg / μl when the specific primers CF01 / CF02 were used only for one PCR amplification.

Embodiment 3

[0073] The sensitivity test of embodiment 3Nested PCR

[0074] Same as Example 2, the DNA of bacterial strain Ceratocystis fagacearum ATCC 200423 was diluted into different concentration gradients, respectively 1 × 10 2 pg / μl, 10pg / μl, 1pg / μl, 1×10 -1 pg / μl, 1×10 -2 pg / μl, take 1 μl as a template, and perform two rounds of amplification, the first round and the second round, in sequence according to step 2 and step 3 of Example 1.

[0075] The electrophoresis results of the products obtained after two rounds of amplification are shown in image 3 , in the figure, M is a 2000-bp DNA marker; 1-5 are amplification products in 25μl reaction system with DNA concentrations of 100pg, 10pg, 1pg, 0.1pg, and 0.01pg; 6 is a negative control. From image 3 It was shown that after two rounds of PCR amplification, the lowest detectable bacterial DNA concentration was 1pg / μl.

[0076] with Example 2 figure 2 In comparison, it can be seen that the Nested PCR of the present invention us...

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Abstract

The present invention relates to a nested PCR detection method of oak wilt which can be carried and transferred by log, wooden packing and the like. The method of the invention designs a pair of specific primers CF01 / CF02 for distinguishing oak wilt, sample DNA is used as template, universal primers ITS1 / ITS4 are adopted to perform the first round PCR amplification, then the product of the first round PCR amplification is used as template to perform the second round PCR amplification of the specific primer, the product of the amplification is detected by electrophoresis test and if 280bp of DNA specific fragments exist, the tested pathogen is detected to be oak wilt. The detection method can be used for detection on the condition of the existence of trace pathogenic bacteria, the sensitivity is 1pg DNA, when the method is used for detecting pathogen in soil, only the existence of one spore of pathogen can realize the detection so that the method has strong reliability and is suitable for the fast oak wilt detection for port nursery stock and wooden packing.

Description

technical field [0001] The invention relates to a method for detecting the fungus of Fusarium wilt mainly carried by logs and wooden packages, in particular to a nested PCR detection method (or Nested PCR method) for the fungus of Fusarium wilt, which belongs to the field of biotechnology. Background technique [0002] Oak wilt is a fungal disease caused by Ceratocystis fagacearum (abbreviated as C. fagacearum), which mainly harms Quercus species. It is currently distributed in the United States in North America, Canada, and Bulgaria, Poland, Romania and other countries in Europe. , has not been found in China. [0003] Oak blight (Ceratocystis fagacearum), first discovered in Wisconsin in 1942, caused the rapid death of the local red oak. Since then, the disease has continued to spread and has been found in more than 20 states in the central and eastern regions of the United States. It has become a major devastating disease of oak tree species in the United States. Thousa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11C12R1/645
Inventor 吴翠萍陈贯源粟寒李彬郑斯竹安榆林叶建仁
Owner ANIMAL AND PLANT & FOOD DETECTION CENTER JIANGSU ENTRY EXIT INSPECTION AND QUARANTINE BUREAU
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