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Real-time fluorescence PCR detection method of Ceratocystis fagacearum (Bretz) Hunt

A real-time fluorescence and detection method technology, applied in the biological field, can solve problems such as unreported, and achieve the effects of strong reliability, shortened quarantine period, and high sensitivity

Inactive Publication Date: 2010-05-05
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

And about the real-time fluorescent PCR (Real-time PCR) detection method of Fusarium wilt bacterium, even other molecular detection methods, all have no report both at home and abroad

Method used

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  • Real-time fluorescence PCR detection method of Ceratocystis fagacearum (Bretz) Hunt
  • Real-time fluorescence PCR detection method of Ceratocystis fagacearum (Bretz) Hunt
  • Real-time fluorescence PCR detection method of Ceratocystis fagacearum (Bretz) Hunt

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Specific detection of Real-time PCR primers of Fusarium oxysporum

[0027] 1. Extract the DNA of the known samples of Fusarium wilt and the hypha DNA of the control strain in Table 1

[0028] 1.1 Strain culture and mycelium collection

[0029] The strain was attached to the PDA medium and cultured in the dark at 20-25°C for 10 days. The hyphae were gently scraped off with an inoculating needle, placed in a centrifuge tube, freeze-dried, and stored at -20°C for later use.

[0030] 1.2 Use the improved CTAB method to extract mycelial DNA, the specific operation is as follows:

[0031] 1) Take 0.5g of frozen mycelium and place it in a 2.0ml flat-bottomed centrifuge tube, add 2 steel balls with a diameter of 3mm, close the tube cap, place it on the Oscillating Mill MM400, and grind for 3 minutes at 30f / min;

[0032] 2) Take out the steel ball, add 500μl CTAB, freeze in liquid nitrogen for 2 minutes, then 75°C water bath for 2 minutes, repeat twice, and finally melt in 75°C w...

Embodiment 2

[0050] Example 2 Real-time PCR sensitivity test

[0051] The DNA of the extracted strain Ceratocystis fagacearum ATCC 200423 was measured on a spectrophotometer and then diluted to 1×10 in a 10-fold gradient. 2 pg / μl, 10pg / μl, 1pg / μl, 1×10 -1 pg / μl, 1×10 -2 pg / μl standard gradient solution, take 1μl as the template, and perform amplification on the ABI 7500Fast fluorescent quantitative PCR instrument according to the conditions of step 2 in Example 1. The results show that figure 1 In the amplification curve, 4 "S" amplification curves can be seen, that is, 4 concentrations of DNA have been amplified, so the lowest detectable DNA concentration of pathogens is 0.1 pg / μl. in figure 2 In the middle, the melting curve Tm=83.5±0.5°C, when it is used to test samples in the future, the possibility of false positives can be ruled out based on this Tm value. In addition, see image 3 , The linear relationship of the standard curve, r 2 =0.997 is very close to 1, which shows that the 10-f...

Embodiment 3

[0052] Example 3 Using Real-time PCR to detect the number of pathogenic spores in soil

[0053] 1.1 Test of spores carrying pathogens in simulated soil

[0054] Add 1×10 to 0.3g sterilized soil 5 Pcs, 1×10 4 Pcs, 1×10 3 Pcs, 1×10 2 A spore suspension of Ceratocystis fagacearum ATCC 200423 with a total of 6 gradients of 1, 10, 1

[0055] 1.2 Extract DNA from soil samples with bacteria

[0056] 1) Take 0.3 g of soil, dry it, and grind it with a grinder;

[0057] 2) Add the ground fine powder into a 2.0ml centrifuge tube, add 500μl of 0.4% (mass / volume) skimmed milk powder solution, vortex and mix well, centrifuge at 12000rpm for 15 minutes;

[0058] 3) Take the supernatant, add an equal volume of proteinase K buffer (50mM Tris-HCl PH=8., 2.5mM EDTAPH=8.0, 1% SDS and 10μg / ml proteinase K), and bathe in water at 55°C for 1-3 hours;

[0059] 4) Add 1 / 2 of the total volume of 7.5M NH 4 AC solution, mix upside down, centrifuge at 12000rpm for 15 minutes;

[0060] 5) Aspirate the supernatant and a...

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Abstract

The invention relates to a real-time fluorescence PCR detection method of Ceratocystis fagacearum (Bretz) Hunt, comprising the following steps: taking sample DNA as a template; carrying out real-time fluorescence PCR augmentation of the Ceratocystis fagacearum (Bretz) Hunt by utilizing a specific primer CF01 / CF02 and fluorescent dye SYBR Green I; pre-augmenting the system for 30 seconds at 95 DEG C in an ABI7500Fast fluorescent quantitative PCR instrument and then 3 seconds at 95 DEG C, extending 25 seconds at 60 DEG C with 40 cycles in all, and synchronously acquiring the fluorescence many times at each cycle extension step; immediately analyzing a fusion curve by the following procedures: 1min at 95 DEG C, 1min at 60 DEG C, keeping 10s for every rise of 0.5 DEG C from 60 DEG C, and continuously rising the temperature 70 times till reaching 95 DEG C and indicating the detected germ sample is the Ceratocystis fagacearum (Bretz) Hunt if an augmentation graph shows a remarkable S-shaped curve, and the Tm absorption peak value in the fusion curve is identical with positive control. The invention has high detection sensitiveness to the Ceratocystis fagacearum (Bretz) Hunt, is convenient and rapid and can be used for monitoring and controlling logs, lignin packages, vegetable materials and the carried soil Ceratocystis fagacearum (Bretz) Hunt at the real time at all quarantine ports.

Description

Technical field [0001] The invention relates to a real-time fluorescent PCR detection method for Quercus fusarium wilt and belongs to the field of biotechnology. Background technique [0002] Oak wilt is a fungal disease caused by the bacterium Ceratocystis fagacearum, which mainly harms Quercus tree species. It is currently distributed in North America, the United States, Canada, and Europe, Bulgaria, Poland, Romania and other countries. It has not been found in China. . [0003] Oak wilt (Ceratocystis fagacearum), first discovered in Wisconsin in 1942, caused rapid death of local red oak. Since then, the disease has continued to spread. At present, it has been found in more than 20 states in the central and eastern regions of the United States, and it has become a major devastating disease for oak species in the United States. Thousands of oak trees die every year in Wisconsin and Minnesota, which are the most harmful. At present, it has been listed as a quarantine pest by the...

Claims

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Application Information

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IPC IPC(8): C12Q1/68G01N21/64C12R1/645
Inventor 吴翠萍陈贯源李彬粟寒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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