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Molecular detection kit and method for detecting trace of bursaphelenchus xylophilus in pine wood

A technology for pine xylophilus and molecular detection, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of false negative test results, inability to effectively amplify ITS fragments, etc., and improve detection accuracy Effect

Pending Publication Date: 2018-08-10
ZHEJIANG FORESTRY ACAD
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Problems solved by technology

However, in the actual operation process, when the pine wood sample is in the early stage of infection and only contains a small amount of B. xylophilus, PCR cannot effectively amplify the ITS fragment of the expected size, resulting in false negative results.

Method used

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  • Molecular detection kit and method for detecting trace of bursaphelenchus xylophilus in pine wood
  • Molecular detection kit and method for detecting trace of bursaphelenchus xylophilus in pine wood
  • Molecular detection kit and method for detecting trace of bursaphelenchus xylophilus in pine wood

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Experimental program
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Effect test

Embodiment 1

[0056] (1) Extraction of pine wood nematode genomic DNA in pine samples: take 16 pieces of pine wood samples to be tested (Table 1), wrap them with gauze (≥4 layers), and put them in a water bath at 37°C overnight (≥12h). High-speed centrifugation (15000rpm / min), the immersion solution of the pine wood sample is concentrated. After centrifugation, most of the supernatant was removed, and 4 ml was reserved for resuspending the pellet. Divide the suspension into two parts, one part is centrifuged thoroughly, and the precipitate is used for DNA extraction; the other part is used for microscope observation with a magnification of 10-20 times.

[0057] Table 1: Pine wood samples tested

[0058]

[0059] (2) The sample obtained in step 1 was thoroughly ground with liquid nitrogen, and the genomic DNA was extracted using the Qiagen DNeasy Blood & Tissue Kit (QIAGEN, USA) kit, and the genomic DNA of the sample was extracted according to the instructions. The obtained genomic DNA ...

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Abstract

The invention relates to a molecular detection kit for detecting trace of bursaphelenchus xylophilus in pine wood, and a method for detecting the bursaphelenchus xylophilus in pine wood by adopting the molecular detection kit. Specific amplification primers comprise a primary PCR specific primer and a nest PCR ITS specific primer, wherein the sequences of the nest PCR ITS specific primer are as follows: the upstream primer: 5'-ATGCGATTGGTGACTTCGGTT-3', and the downstream primer: 5'-CCCGCAAGGCTTCACGACGA-3'. The molecular detection kit and the method have the beneficial effects that the kit candetect trace of bursaphelenchus xylophilus from pine wood infected with diseases at the early stage, so that the judgment of the false negative result is avoided, and the detection accuracy for the trace of B. xylophilus in the pine wood is obviously improved.

Description

(1) Technical field [0001] The invention relates to a molecular detection kit for trace pine wood nematode (Bursaphelenchus xylophilus) in pine wood and a method for detecting pine wood nematode in pine wood by using the molecular detection kit. (2) Background technology [0002] Pine wood nematode [Bursaphelenchus xylophilus (Steiner&Buhrer) Nickle], belonging to Animalia, Nematoda, Secernentea, Tylenchida, Apelenchoididea, Bursaphelenchus ), is the pathogen of pine wilt disease (Pine Wilt Disease, PWD), which can cause a devastating blow to pine, and its control is extremely difficult. It is an internationally recognized quarantine pest and a major forestry alien pest in my country. The disease has spread rapidly in my country since it was first discovered in Zijin Mountain, Nanjing, my country in 1982. At present, the epidemic is still expanding, causing serious losses to my country's economy, society and ecology. [0003] A key measure for the integrated control of pine ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6858C12Q1/6848
CPCC12Q1/6848C12Q1/6858C12Q1/6888C12Q2600/156C12Q2531/113C12Q2549/119
Inventor 叶碧欢李海波陈友吾胡杨胡传久李楠魏海龙杜国坚宋其岩沈建军朱玲琴
Owner ZHEJIANG FORESTRY ACAD
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