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

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...

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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 clearly distinguish B. xylophilus from B. xylophilus;

[0006] 3. Although DNA hybridization technology can accurately and quickly identify pine xylophilus, the technology is difficult, expensive, and has radioactive hazards, which is not conducive to large-scale promotion;

[0007] 4. Although the random amplified polymorphic DNA technique (RAPD) has the advantages of rapidity, simplicity, sensitivity and no need for radioactive isotope labeling, it also has the disadvantages of complex maps, poor repeatability and comparability. limitations

Bai Gang et al. (2005 Bai Gang, Li Haiyan, Ma Hongzhou et al. Immunological detection method of pine wood nematode antigen on cut surface of infected wood. Forestry Science, 2005, 41(6): 101-104) using anti-pine wood nematode serum, established in The method of directly performing immunohistochemical staining on the cut surface of infected wood to detect pine xylophilus antigens, but the operation is complicated, and factors such as antibody specificity have brought certain difficulties to practical application; Wang Yanhui (2007 Wang Yanhui. and detection technology research. Guangzhou: South China Agricultural University, 2007.30-36) By designing and combining peripheral general primers and specific primers, using nested PCR technology to effectively detect B. nematodes, but the steps are cumbersome and add many uncertain factors, it is still in the research and development stage

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