Primer and method for detecting specificity of pine wood nematode in Chinese red pine died wood of illness, and application thereof

A technology of pine wood nematode and detection method, which is applied in the field of genetic detection of pine wood nematode, can solve problems such as high technical difficulty, difficult determination of separation results, and increase of uncertain factors, so as to improve detection efficiency, save separation time, Detect quick effects

Inactive Publication Date: 2009-11-25
SOUTH CHINA AGRI UNIV
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Problems solved by technology

[0004] 1. The morphological identification of pine wood nematode is simple and easy, and it is the current routine method for nematode species identification. However, the nematode is small in shape and needs to be observed by professionals under a microscope. The morphological characteristics of B. xylophilus, P. spp., P. colimoides, B. xylophilus and B. xylophilus are similar to those of B. xylophilus, which also makes morphological identification more difficult. In addition, the appearance and symptoms of plants in the early stage of infection are not obvious, which means that It also makes the early diagnosis of pine wood nematode difficult. It is not always possible to isolate the adult pine wood nematode from the diseased pine wood, and most of the pine wood nematodes exist in the form of larvae. It takes time to cultivate the adults. Diagnosis delayed the best prevention and treatment period;
[0005] 2. Protein electrophoresis analysis is a molecular technique that was earlier applied to the study of nematode taxonomy. Many scholars have carried out studies on the difference of isozymes between B. xylophilus and B. xylophilus strains. However, the isozyme technique requires a lot of A clear electrophoretic pattern can only be obtained from the sample, and the expression of the protein is related to the environment and the growth and development stage of the nematode. So far, there is no isozyme that can...

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  • Primer and method for detecting specificity of pine wood nematode in Chinese red pine died wood of illness, and application thereof
  • Primer and method for detecting specificity of pine wood nematode in Chinese red pine died wood of illness, and application thereof
  • Primer and method for detecting specificity of pine wood nematode in Chinese red pine died wood of illness, and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Embodiment 1 Primer specificity test of the present invention

[0052] 1. Prepare primers: Synthesize the specific detection primers of pine xylophilus in the diseased and dead wood of Pinus massoniana by Gene Synthesis Company, the nucleotide sequence of its upstream primer is as shown in SEQ ID NO: 1, and the nucleotide sequence of the downstream primer is as shown in SEQ ID NO: 1. ID NO: 2. Primers were stored in a -20 refrigerator.

[0053] 2. PCR amplification

[0054] PCR amplification reaction system: 10×PCR buffer 2.5μl (Mg 2+ free), dNTP (2.5mmol L -1 )2 μl, Mg 2+ (25mmol·L -1 ) 3 μl, each of primers P1 (10 μM) and P2 (10 μM) 1 μl, Taq DNA polymerase 5U·μl -1 0.125 μl, template DNA 2 μl, and sterilized double distilled water to make up to a total volume of 25 μl.

[0055] PCR amplification reaction conditions: pre-denaturation at 94°C for 3min; denaturation at 94°C for 45s, annealing at 53°C for 30s, extension at 72°C for 1min, after 35 cycles, and fina...

Embodiment 2

[0072] Example 2 Preparation of DNA template from diseased and dead wood of Pinus massoniana

[0073] This example optimizes the amount of WLB and proteinase K required for the preparation of DNA templates from dead wood of masson pine.

[0074] The DNA template was prepared by using the diseased and dead wood of Pine masson. If 2 mg of the dead wood of Masson pine was used, it was difficult to amplify the target fragment, so 5 mg of the dead wood of Masson pine was used for the test.

[0075] Prepare WLB lysate, the formula is: containing 2.5mmol L -1 Dithiothreitol, 1.125% Tween-20, 0.025% gelatin, 125mmol L -1 KCl, 25mmolL -1 Tris-HCl (pH 8.0) and 3.75mmol L -1 MgCl 2 .

[0076] Proteinase K is commercially available.

[0077] In this example, three kinds of lysates are used: (1) 32 μL of WLB lysate and 4 μL of proteinase K; (2) 100 μL of WLB and 8 μL of proteinase K; (3) 200 μL of WLB and 10 μL of proteinase K. Prepared operations:

[0078] Put 5 mg of dead wood...

Embodiment 3

[0080] Example 3 Optimization of PCR amplification conditions

[0081] In this embodiment, the annealing temperature and magnesium ion concentration in PCR amplification are optimized.

[0082] 1. Optimization of annealing temperature

[0083] Using 47°C, 47.6°C, 48.2°C, 49.4°C, 50.8°C, 52.2°C, 53.7°C, 55.1°C, 56.6°C, 57.8°C, 58.4°C and 59°C as temperature gradients, the genomic DNA of pine xylophilus was amplified by PCR , PCR amplification results such as Figure 6 As shown, clear bands can be amplified at the 12 tested temperatures, and the PCR yields are the largest at 52.2°C and 53.7°C, so the annealing temperature of the present invention is preferably 53°C.

[0084] 2. Optimization of magnesium ions

[0085] In the 25μL PCR amplification reaction system, take 1.5mmolL -1 , 2.0mmolL -1 , 2.5mmolL -1 , 3.0mmolL -1 , 3.5mmolL -1 and 4.0mmolL -1 A total of 6 gradients of magnesium ion concentration were used to perform PCR amplification on the genomic DNA of pine x...

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Abstract

The present invention discloses a primer and a method for detecting a specificity of a pine wood nematode in a Chinese red pine died wood of an illness, and an application thereof, wherein a nucleotide sequence of an upstream primer of the specificity detection primer is represented by SEQID NO:1, a nucleotide sequence of a downstream primer is represented as SEQ ID NO:2. the primer of the invention is capable of separating the pine wood nematode from other 12 nematodes of the Chinese red pine fat wood and Chinese red pine as well as common four fungus and ten bacteria of the Chinese red pine. the invention also provides a method for implementing a PCR amplification of the pine wood nematode of the diseased wood directly without separation of the pine wood nematode from the diseased wood, after rubbing the diseased wood liquid nitrogen, by means of two cleavages of WLB and protease K, a DNA formwork for the PCR amplification will be obtained after purifying, the amplification efficiency will achieve 100%, a separation time is saved and a detection efficiency is improved. The detection method of the invention is simple and high-efficiency, has a high accuracy and the specificity; it is capable of being produced into reagent kits to popularize widely.

Description

technical field [0001] The invention relates to genetic detection of pine wood nematode, in particular to a specific detection primer, detection method and application of pine wood nematode in diseased and dead wood of masson pine. Background technique [0002] Pine wood nematode disease is an important quarantine disease, and the current national occurrence area of ​​pine wood nematode disease is 87,000 hm 2 , the cumulative number of dead pine trees is more than 35 million. The diseased trees in the epidemic area not only cannot be used normally, but also affect the circulation of other agricultural and sideline products and forest products, causing direct economic losses of 2.5 billion yuan and indirect economic losses of 25 billion yuan. Ecological environment, foreign trade exports The social and economic development has been seriously affected, and it has been included in the internal and external quarantine objects of our country. [0003] Regarding the identificatio...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 王新荣胡月清孔祥超贾文慧朱孝伟钟填奎纪春艳
Owner SOUTH CHINA AGRI UNIV
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