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PCR primer, PCR detection kit and detection method for detecting B.xylophilus

A technology of pine wood nematode and detection method, applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., capable of solving expensive, high-cost, radioactive and other problems

Inactive Publication Date: 2021-11-26
BEIJING FORESTRY UNIVERSITY
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  • 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 is poor in repeatability; RFLP technology distinguishes B. xylophilus from P. Nematode endonucleases are expensive and time-consuming; fluorescent PCR is sensitive and fast, but expensive; PCR technology is easy to operate and less expensive, and the premise is that PCR primers with strong specificity need to be screened

Method used

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  • PCR primer, PCR detection kit and detection method for detecting B.xylophilus
  • PCR primer, PCR detection kit and detection method for detecting B.xylophilus
  • PCR primer, PCR detection kit and detection method for detecting B.xylophilus

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Embodiment 1

[0029] Embodiment 1 detects the design of the PCR primer of pine xylophilus, screening and the establishment of detection method

[0030] 1. Materials and methods

[0031] 1.1 Test material

[0032] 1.1.1 Biomaterials

[0033] Monochamus monochamus and Spruce monochamus collected in the field from 2017 to 2019 (Table 1); B. xylophilus and B. xylophilus cultivated in the laboratory (Table 2).

[0034] Table 1 Monochamus monochamus and Spruce monochamus collected in the field

[0035]

[0036] Table 2 B. xylophilus and B. xylophilus cultivated in the laboratory

[0037]

[0038]

[0039] 1.1.2 Chemical reagents

[0040] Liquid nitrogen; TE buffer solution; PrimeSTAR HS DNA (Premix) (TaKaRa).

[0041] 1.2PCR amplification

[0042] 1.2.1 PCR primer design

[0043] Using the Bx-cpi gene as the target gene, primers specific to B. xylophilus were designed by Primer 6.0. 1.2.2 Establishment of PCR reaction system

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Abstract

The invention discloses a PCR primer, a PCR detection kit and a detection method for detecting B.xylophilus. A PCR primer pair capable of specifically amplifying B.xylophilus is designed and screened by taking a conserved Bx-cpi gene in a B.xylophilus species as a target gene, and the PCR primer pair consists of two primers with nucleotide sequences shown as SEQ ID No.9 and SEQ ID No.10. According to the invention, the specific PCR primer pair is further utilized to establish a PCR detection method for B.xylophilus. The method can rapidly detect whether a sample to be detected carries the B.xylophilus. or not, and the detection method has the advantages of strong specificity, high accuracy, simple operation, short time consumption, low cost and the like. The method has application value for monitoring occurrence and spreading of the Pine Wilt Disease, overall early warning and control strategies of the Pine Wilt Disease and the like.

Description

technical field [0001] The present invention relates to the molecular detection of pine wood nematode, in particular to a PCR primer set and a PCR detection kit for detecting pine wood nematode, and also relates to the application of the PCR primer set and PCR detection kit in detecting pine wood nematode, belonging to The field of molecular detection of pine xylophilus. Background technique [0002] Pine wilt disease, also known as pine wilt disease (Pine Wilt Disease, PWD), is caused by the pathogenic pine wood nematode (Bursaphelenchus xylophilus (Steiner&Buhrer) Nickle) (Nickle WR, Golden AM, MamiyaY, et al. On the taxonomy and morphology of The pine wood nematode, Bursaphelenchus xylophilus (Steiner & Buhrer 1934) Nickle 1970. Journal of Nematology, 1981, 13:385-392) caused a devastating pine forest disease. [0003] Pine wood nematode disease is a kind of pine wood nematode (B.xylophilus) as the pathogen, monochamus spp. (Monochamus spp.) insect (Coleoptera: Cerambyci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
Inventor 孟繁丽理永霞张星耀
Owner BEIJING FORESTRY UNIVERSITY
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