Kit for qualitative detection of pinewood nematode and detection method thereof

A pine wood nematode, qualitative detection technology, applied in the direction of microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of expensive instruments, achieve good repeatability, good practicability, and no significant difference

Active Publication Date: 2012-12-19
NANJING FORESTRY UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method directly extracts the DNA of pine wood nematode in the diseased wood, combined with PCR technology, can detect 1 pine wood ne

Method used

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  • Kit for qualitative detection of pinewood nematode and detection method thereof
  • Kit for qualitative detection of pinewood nematode and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The kit for qualitative detection of pine xylophilus consists of the following:

[0031] (1) DNA extraction reagent: It can be a conventional commercially available DNA extraction reagent, preferably using the instrument-free nucleic acid extraction kit from Hangzhou Ustar Biotechnology Co., Ltd.

[0032] (2) Pine xylophilus nucleic acid constant temperature amplification reaction solution: two peripheral primers (0.1 μmol each), two probes (0.5 μmol each), two cross primers (0.5 μmol each), 10×Thermol buffer, MgSO 4 (6mmol), dNTPs solution (0.4mmol), Bst DNA polymerase (10U), sterile double distilled water to make up 16μL.

[0033]Among them: the sequence of the forward peripheral primer is 5'-TCCTCACCTGGCTCTT-3'; the sequence of the reverse peripheral primer is '-CTAAACTCCCCCATCTC-3'; the sequences of the two probes are respectively: forward 5' end Biotin labeled probe 5-biotin - TCTTTTCGGCCACACC-3', forward 3'-end fluorescein isothiocyanate (FitC) labeled probe 5-AG...

Embodiment 2

[0040] Qualitative detection of pine wood nematode with the kit of embodiment 1, specific method is:

[0041] (1) Use a DNA extraction kit to extract DNA from the specimen to be tested. For the specific protocol, please refer to the instructions of the DNA extraction kit.

[0042] (2) Take 4 μL of sample DNA as a template and add it to a PCR tube containing 16 μL of pine wood nematode nucleic acid constant temperature amplification reaction solution. Add equal volumes of positive control template and negative control template to the control PCR tube, and perform amplification at 63°C. Increase reaction 60min.

[0043] (3) Put the reacted PCR tube into the nucleic acid anti-pollution detection device (test strip, see CN1888902A or CN1811447A) for detection, and interpret the result after 15 minutes. When the sample contains pine wood nematode nucleic acid, the detection line of the test strip is positive.

[0044] The experiment was repeated 3 times, and there was no signifi...

Embodiment 3

[0046] The specificity of B. xylophilus was detected with the kit of Example 1.

[0047] Detect pine wood nematode, pseudo pine wood nematode and positive control substance according to the method of embodiment 2; The result is as follows figure 1 . From figure 1 It can be seen from the test results that the detection of pine wood nematode nucleic acid by the kit of the present invention has strong specificity.

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Abstract

The invention discloses a kit for qualitative detection of pinewood nematode and a detection method thereof. Reagents of the kit comprise a DNA extract, a reaction liquid of constant-temperature amplification of pinewood nematode nucleic acid, a positive control and a negative control, wherein the positive control is a plasmid containing a DNA fragment of a pinewood nematode BxS1 gene, and the negative control is sterile double distilled water. The kit detects the pinewood nematode through constant-temperature amplification of nucleic acids, and sensitivity thereof is that 100 copies can be detected in each reaction system. Repetitive experiments show that detection results have no significant differences and are good in repeatability. Only 2 hours are required to finish the sample detection, thereby greatly reducing detection time. Meanwhile, only one person is needed to finish all operation process of the kit, and one to hundreds of samples can be detected for one time, thereby reducing manpower waste, meeting requirements of rapid detection of the pinewood nematode and thus being quite practical.

Description

technical field [0001] The invention relates to a detection method of pine wood nematode, in particular to a kit for qualitative detection of pine wood nematode and a detection method thereof. Background technique [0002] Pine wood nematode is also called pine wilt disease. is a devastating epidemic of pine trees. In my country Monochamus alternatus ( Monochamus alternatus ) is its main media insect. The pathogenicity is strong, and the host dies quickly; once it occurs, it is difficult to control. Pine trees infected with pine xylophilus can die within 40 days, and it only takes 3-5 years for the affected stands to be destroyed from the onset of pine trees to the destruction of the entire pine forest, and the spread spreads rapidly. It not only causes huge losses to the national economy, but also destroys the natural landscape and ecological environment, and poses a serious threat to my country's rich pine forest resources. Since its discovery in 1982, it has spread r...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 叶建仁胡林陈凤毛侯建华吴小芹孙波黄麟詹国辉徐高连贺君丽范椰吴芳
Owner NANJING FORESTRY UNIV
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