LAMP (Loop-mediated Isothermal Amplification) primer group, kit and detection method for detecting bursaphelenchus xylophilus nickle

A detection kit and pine wood nematode technology are applied in the molecular detection of pine wood nematodes carried by pine wood nematodes, and the molecular detection of pine wood nematodes can solve the problems of expensive, radioactive, and expensive endonucleases.

Active Publication Date: 2017-08-08
INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, although DNA probe technology can effectively identify B. xylophilus, it is expensive and radioactive; RAPD technology analyzes the relationship between B. xylophilus and related species, and the result has poor repeatability; RFLP technology distinguishes B. xylophilus from P. Styloid nematodes, endonucleases are expensive and time-consuming; fluorescent PCR is sensitive and fast, but expensive; PCR technology is easy to operate, less expensive, but takes a long time

Method used

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  • LAMP (Loop-mediated Isothermal Amplification) primer group, kit and detection method for detecting bursaphelenchus xylophilus nickle
  • LAMP (Loop-mediated Isothermal Amplification) primer group, kit and detection method for detecting bursaphelenchus xylophilus nickle
  • LAMP (Loop-mediated Isothermal Amplification) primer group, kit and detection method for detecting bursaphelenchus xylophilus nickle

Examples

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Embodiment 1

[0038] Example 1 Establishment of a LAMP rapid detection method for Monochamus alternatus carrying pine xylophilus

[0039] 1. Materials and methods

[0040] 1.1 Extraction of Monochamus alternatus DNA

[0041] Take 0.2 g of head and thorax tissue of Monochamus alternatus adults, put it into a 2 mL EP tube, put it into a steel ball mill and grind it for 5 min (60 Hz); add 600 μL of 3M guanidine isothiocyanate extraction buffer, lyse at room temperature for 1 h, and mix it upside down during the period. Mix well; add 600 μL Tris-phenol chloroform isoamyl alcohol (25:24:1), centrifuge at 12000 r / min for 10 min; absorb the supernatant, add 600 μL chloroform isoamyl alcohol (24:1), centrifuge at 12000 r / min for 10 min; absorb the supernatant Add 600 μL of isopropanol to precipitate at -20°C for 20 minutes, centrifuge at 12,000 r / min for 10 minutes; discard the supernatant, wash the precipitate twice with 70% ethanol, and centrifuge at 12,000 r / min for 3 minutes each time; blot th...

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Abstract

The invention discloses a LAMP (Loop-mediated Isothermal Amplification) primer group, a kit and a detection method for detecting bursaphelenchus xylophilus nickle. According to the invention, firstly, a conserved SYG-2 gene in the bursaphelenchus xylophilus nickle is used as a target gene, the LAMP primer group with high specificity for bursaphelenchus xylophilus nickle is designed and screened out, and the LAMP primer group can specifically distinguish bursaphelenchus xylophilus nickle from bursaphelenchus mucronatus. The invention utilizes the LAMP primer group to establish the bursaphelenchus xylophilus nickle LAMP rapid detection kit. The invention further utilizes the LAMP primer group to establish the bursaphelenchus xylophilus nickle LAMP rapid detection method. According to the method, whether bursaphelenchus xylophilus nickle is carried in a sample or not can be accurately distinguished. The LAMP detection method disclosed by the invention is high in specificity, simple and convenient to operate and short in time consumption, and has the important significance for rapid detection on bursaphelenchus xylophilus nickle carried in monochamus alternatus and total early warning and a control strategy for the pine wilt disease.

Description

technical field [0001] The present invention relates to the molecular detection of pine wood nematodes, in particular to a LAMP primer set and a kit for detecting the pine wood nematode carried by Monochamus alternata, and also to the LAMP primer set and the kit for detecting the pine wood nematode carried by Monochamus alternata. The application in nematodes belongs to the molecular detection field of monochamus monochamus carrying pine xylophilus. Background technique [0002] Pine wilt disease, also known as pine wilt disease and fusarium wilt, is caused by the pine wood nematode [Bursaphelenchus xylophilus (Steiner&Buhrer) Nickle], mainly through insects of the genus Monochamus on pine trees. Natural transmission by feeding and spawning, and long-distance transmission by transportation of diseased wood and diseased wood processed wood. The spread of pine xylophilus in the forest area is mainly by Monochamus alternatus, which can damage Pinus spp., Larix spp., Picea spp....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2531/119
Inventor 汪来发王曦茁朴春根李永林彩丽薛寒
Owner INST OF FOREST ECOLOGY ENVIRONMENT & PROTECTION CHINESE ACAD OF FORESTRY
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