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Pine wood nematode pcr detection kit and its detection method

A detection kit and technology of pine wood nematode, which is applied in the field of microbial detection, can solve the problems that it is difficult to meet the rapid quarantine requirements of forest plant quarantine checkpoints, the detection accuracy needs to be improved, and the detection specificity is not strong, and the results are objective and reliable. DNA Good extraction effect and high specificity

Active Publication Date: 2017-06-30
苏州天隆生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first category is common PCR detection methods, such as the "pine wood nematode detection kit and its detection method" with the patent application number 200310106109.5. In actual production; the second type is real-time fluorescent PCR detection method, such as "a method for detecting pine wood nematode and its special primers and probes" with the patent application number 200310109374.9, etc., these detection methods more or less exist to detect The specificity is not strong, and the detection accuracy needs to be improved, so it has not been applied in production practice; the third category is the constant temperature amplification detection technology, such as the patent No. 201210303532.3 "A kit for qualitative detection of pine wood nematode and Its detection method”, this detection technology has yet to be verified in production
[0004] In summary, the above-mentioned molecular detection methods of pine wood nematodes are difficult to meet the rapid quarantine requirements of various forest plant quarantine inspection stations
From the perspective of rapid quarantine, the above-mentioned pine wood nematode detection technologies and methods still have a certain gap from rapid quarantine. In order to meet the rapid identification requirements of quarantine practice, it is necessary to develop more simple, rapid and efficient detection and identification technologies and methods.

Method used

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  • Pine wood nematode pcr detection kit and its detection method
  • Pine wood nematode pcr detection kit and its detection method
  • Pine wood nematode pcr detection kit and its detection method

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Experimental program
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Effect test

Embodiment 1

[0058] The raw material of kit of the present invention and preparation:

[0059] 1) Tris: analytically pure, content 99.7%, infrared qualified, pH (5% water) 10.3-10.9, moisture 0.3%, melting point 167-171°C, qualified absorption system, qualified maximum impurity content.

[0060] 2) MgCl 2 : Analytical pure, the content is not less than 99%, the aqueous solution reaction is qualified, and the highest impurity content is qualified.

[0061] 3) EDTA: analytically pure, white crystalline powder, soluble in water, the solution is acidic, insoluble in alcohol, the content is not less than 99.5%, the aqueous solution reaction is qualified, the complexing force test is qualified, and the highest content of impurities is qualified.

[0062] 4) HCl: analytically pure.

[0063] 5) Purified water: pure water, treated by a pure water machine, with a resistivity of 18.2MΩ.

[0064] 6) Synthetic primers and probes:

[0065] Conserved sequence of pine xylophilus exotoxin A gene, that ...

Embodiment 2

[0099] 1. Reagent preparation:

[0100] 1. Take the kit out from -20°C and equilibrate at room temperature for 20 minutes.

[0101] 2. Centrifuge the hot-start Taq enzyme and UNG enzyme briefly, and put them at -20°C for later use.

[0102] 3. After determining the number n of reaction tubes to be carried out, take out the PCR reaction solution of pine wood nematode, add n×44.3 μl pine wood nematode PCR reaction solution, n×0.5 μl hot-start Taq enzyme and n×0.2 μl UNG enzyme to one Put it in a centrifuge tube and vortex to mix well. After instantaneous centrifugation, dispense 45 μl into each PCR reaction tube, cover the tube cap and store in a 4°C refrigerator in the dark for later use.

[0103] 4. Put the positive control, negative control, and internal reference in a 4°C refrigerator for later use.

[0104] 2. Processing of samples to be tested:

[0105] Applicable specimen type: wound swab

[0106] 1. Add 1ml of normal saline to the 1.5ml centrifuge tube, fully wash th...

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Abstract

The invention discloses a pine wood nematode PCR (polymerase chain reaction) detection kit. The detection kit comprises a PCR reaction solution containing a primer A and a fluorescent probe A, wherein the primer A is divided into an upstream primer A and a downstream primer A; the nucleotide sequence of the upstream primer A is 5'-CCGTTTGGTGATGTTGTTTCA-3'; the nucleotide sequence of the downstream primer A is 5'-GCAAGGCTTCACGACGAA-3'; and the nucleotide sequence of the fluorescent probe A is 5'-FAM-CTCAACAAACCGCAGCCAGGACTC-BHQ-3'. The invention further discloses a pine wood nematode PCR detection method. The detection kit disclosed by the invention has the advantages of good DNA (deoxyribonucleic acid) extraction effect, simple operation, short consumed time, objective and reliable results, high sensitivity, high specificity and the like.

Description

technical field [0001] The invention relates to the technical field of microbial detection, in particular to a PCR detection kit for pine xylophilus and a detection method thereof. Background technique [0002] Pine wood nematode (North American pinewood nematode; Bursaphelenchus xylophilus), the nematode belongs to the phylum Vermiformes, class Nematodes, orders Osmocylinda, Superfamily Slipperidae, and the genus Umbris. The body of nematode adults is about 1 mm long, the tail of the female is nearly conical, with a rounded end; the tail of the male is like a bird's claw, curved towards the ventral surface. Pine wilt, also known as pine blight, is a devastating pest. It is spread in pine trees by vector insects such as Monochamus alternatus, thus causing pine tree diseases. After being infected by pine wood nematode, the needle leaves are yellowish brown or reddish brown, wilting and drooping, the resin secretion stops, and the long beetle intrusion holes or traces of spa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/686C12Q2561/101C12Q2545/101
Inventor 苗保刚彭年才李明李红东倪晓龙李政
Owner 苏州天隆生物科技有限公司
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