Pinewood nematoda detection reagent-box and detection method

Abstract

Two pairs of primer are designed: Pl / P3 is an idiocratic primer for pine wood nematode; P2 / P3 an idiocratic primer for quasi pine wood nematode. The kit includes 1.25ml solution I,1ml solution II, 100U tag enzyme, and 50ul for each contrapositive DNA of wireworm. The solution I is as following constitutes: Worm Lysis Buffer, 500mM KCl, 100mMTris-ClpH 8.0, 15mM MgCl2, 10mMDTT, 4.5% Tween20, 0.1%gelatin, 0.2ug / ul protease K. The solution II is as following constitutes: 2xPCR reaction Buffer, 4.0mM MgCl2, 0.2mM dNTP, 0.8 mu MP1, P2, P3. The invented kit possesses high specificity, sensitiveness and stability, providing fast, sensitive and specific technical method.

Description

(1) Technical field [0001] The invention relates to a pine wood nematode (Bursaphelenchus xylophilus) detection kit and a detection method thereof, belonging to the field of biotechnology. It is specially used for the high-sensitivity and rapid detection of pine wood nematodes carried by customs inbound and outbound timber and wooden packaging. At the same time, it can be used for the detection and diagnosis of pine wood nematode disease. (2) Background technology [0002] Pine wood nematode disease, also known as pine wilt disease, is a devastating disease in forestry, known as forest cancer. Infected pine trees are infected by it, and when the environmental conditions are suitable, the whole plant dies in only about one month. The disease was first discovered in Nagasaki, Kyushu, Japan in 1905, and then reported in the United States, Canada, Mexico, South Korea, Portugal and other places. The pathogenic nematode of the disease is pine wood nematode [Bursaphelenchus xylo...

AI Technical Summary

Problems solved by technology

At present, in ports, the main quarantine technique for pine xylophilus is morphological observation, but morphological observation has the following disadvantages: First, the pine wood nematode intercepted at the port generally exists in the wooden packaging board or backing board of the goods, These materials are generally relatively dry, and the number of nematodes in them is small, and most of them are larvae, which are often in a dormant stage. Their stylets and esophagus have degenerated, and it is difficult to accurately identify them.

However, further cultivation is required to obtain adult worms for identification. This process takes at least 7-10 days, which cannot meet the requirements of rapid quarantine at ports.

Second, some morphological identification features, such as the length of the tail tip of the female, the shape of the tail, and the shape of the mating umbrella of the male, overlap with B. xylophilus, which brings certain difficulties to the morphological identification and easily leads to false detection and missed detection. Phenomenon

[0003] In recent years, with the development of biochemical and molecular biology techniques, some scholars at home and abroad have successively used isoenzyme electrophoresis and PCR-ITS-RFLP techniques to detect and identify pine wood nematodes, but these techniques are limited by the number of nematode populations, nematode Whether the population is single or not, the nematodes intercepted in the port must be purified and cultivated, which is time-consuming and laborious, and it is difficult to meet the requirements of rapid detection

[0004] With the increase of my country's imports of wood and wood packaging goods, the chances of pine wood nematodes entering my country from epidemic areas are also increasing, but my country's ports lack the necessary methods for rapid detection of the pathogen

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