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Nested PCR detection kit for pine wood nematode disease and use method thereof

A detection kit and technology for pine wood nematode disease, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve problems such as false positives, misleading test result judgment, non-specific amplification of PCR detection, etc., to overcome Effects of poor stability, improved sensitivity and specificity, and improved accuracy

Active Publication Date: 2021-08-06
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, non-specific amplification and false positives often occur in conventional PCR detection, which mislead people to judge the test results.

Method used

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  • Nested PCR detection kit for pine wood nematode disease and use method thereof
  • Nested PCR detection kit for pine wood nematode disease and use method thereof
  • Nested PCR detection kit for pine wood nematode disease and use method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] figure 1 It is the implementation diagram of nested PCR detection technology for pine wood nematode (Bursaphelenchus xylophilus).

[0037] Main reagents: DNA extraction kit, recovery kit and plasmid extraction kit were purchased from Tiangen Biochemical Technology Co., Ltd. Primer synthesis and PCR product sequencing were completed by Chengdu Qingke Zixi Biotechnology Co., Ltd.

[0038] 1. Design of PCR primers for rapid molecular detection of pine wood nematode (Bursaphelenchus xylophilus)

[0039] See Table 1 for the source and name of the tested nematodes.

[0040] Table 1 Bursaphelenchus xylophilus and other tested strains

[0041] Numbering Insect species source 1 B. xylophilus Chinese Academy of Forestry Sciences 2 B. xylophilus Dazhou City, Sichuan 3 B. xylophilus Leshan City, Sichuan 4 B. mucronatus Dazhou City, Sichuan 5 B. Hofmanni Yibin City, Sichuan 6 B. rainulfi Xichang City, Sichuan 7 B....

Embodiment 2

[0072] Embodiment 2 builds the nested PCR detection kit of pine wood nematode (Bursaphelenchus xylophilus)

[0073] 1. The components of the nested PCR detection kit for pine wood nematode (Bursaphelenchus xylophilus) include:

[0074] (1) The first round of PCR amplification system (25 μL): TSINGKE Gold Mix (green) 22 μL, 10 mmol / L TLP-W-S 1.0 μL, 10 mmol / L TLP-W-A 1.0 μL, plus B. xylophilus or mixed nematode genome 1.0 μL of DNA extraction solution was used to form the reaction mixture;

[0075] (2) The second round of PCR amplification system (25 μL): 22 μL of TSINGKE Gold Mix (green), 10 mmol / L TLP-N-S 1.0 μL, 10 mmol / L TLP-N-A 1.0 μL, the product was diluted after the first round of PCR reaction Take 1.0 μL at 100 times.

[0076] (3) Lysis solution (solution A);

[0077] (4) Proteinase K (solution B);

[0078] (5) Positive control Y solution;

[0079] The 941bp target band was cut and recovered, the target DNA fragment was cloned into Escherichia coli JM109, the corr...

Embodiment 3

[0092] Example 3 The nested PCR detection kit of pine wood nematode (Bursaphelenchus xylophilus) detects wild diseased pine trees

[0093] 1. Isolation of nematodes

[0094] Cut the quarantine wood section into small pieces with a hatchet, use the improved Bellman funnel separation method, spread double-layer filter paper on the funnel, put the split wood pieces, connect a section of latex tube under the funnel, and clamp it with a water-stop clip Hold the end. Warm water (20-30°C) was added to soak the wood chips, and after 8h-12h, the water-stop clip was opened and water was poured into a 10ml centrifuge tube. After centrifugation, the supernatant was removed with a pipette gun to obtain a large number of nematodes.

[0095] 2. Genomic DNA extraction

[0096] Use a pipette gun to draw single or multiple nematodes into a 2mL centrifuge tube, make up to 100μL with ddH2O, add an equal amount of lysate, 20μL of proteinase K, put in a water bath at 65°C for 30min, denature at 9...

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Abstract

The invention discloses a nested PCR detection kit for pine wood nematode disease and a use method thereof. The kit comprises (1) TSINGKE gold brand Mix (green); (2) primers: 10mmol / L TLP-W-S, 10mmol / L TLP-W-A, 10mmol / L TLP-N-S and 10mmol / L TLP-N-A, wherein the sequence of the primer TLP-W-S is 5'AGACCCTCATTCTTGCC-3', the sequence of the primer TLP-W-A is 5'-GGACAGTAGATGACGTAGAAT-3', the sequence of the primer TLP-N-S is 5'-CGGTATCGGATGTGCTATT-3', and the sequence of the primer TLP-N-A is 5'-CAAGGACAGTAGATGACGTAGAAT-3'; (3) lysate (A liquid); (4) protease K (B liquid); (5) ddH2O; and (6) positive control Y liquid. The kit can quickly and accurately identify pine wood nematode molecules and early diagnose the pine wood nematode disease, and has great significance for controlling quarantine of the pine wood nematode disease and preventing and treating diseases.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a nested PCR detection kit for pine wood nematode disease and a use method thereof. Background technique [0002] Invasive tree pests are increasingly threatening forest ecosystems. Understanding and controlling their spread represents a major challenge for managers, researchers and policy makers. Pine wood nematode disease, namely pine wilt disease, is a very harmful invasive alien species, which is mainly transmitted by the monochamus monochamus. It is one of the four major forest diseases in the world today. It is called the "cancer" of pine trees. The pathogen is pine wood nematode, which belongs to the second-level quarantine object in my country. After pine wood nematodes invade pine trees for 30-90 days, the pine trees will be eaten by pine wood nematodes due to the rapid reproduction of nematodes, resulting in blockage of the wood pith. Transpiration will gradually lose its f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6848
CPCC12Q1/6888C12Q1/6848C12Q2531/113C12Q2549/119C12Q2565/125
Inventor 刘应高刘广川韩珊
Owner SICHUAN AGRI UNIV
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