Real-time fluorescent PCR (polymerase chain reaction) detection method for dendroctonus pseudotsugae as well as primers and probe for detection

A detection method, real-time fluorescence technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc.

Pending Publication Date: 2021-06-08
海口海关热带植物隔离检疫中心
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Problems solved by technology

However, there is no real-time fluorescent PCR rapid identifica...

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  • Real-time fluorescent PCR (polymerase chain reaction) detection method for dendroctonus pseudotsugae as well as primers and probe for detection
  • Real-time fluorescent PCR (polymerase chain reaction) detection method for dendroctonus pseudotsugae as well as primers and probe for detection
  • Real-time fluorescent PCR (polymerase chain reaction) detection method for dendroctonus pseudotsugae as well as primers and probe for detection

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Embodiment Construction

[0039] In the following, the technical solution of the present invention will be further explained in conjunction with the accompanying drawings, however, the present invention is not limited to the following embodiments.

[0040] A kind of real-time fluorescent PCR detection method of Douglas fir beetle, specifically comprises the following steps:

[0041] (1) Design primers and probes

[0042] The following is the process of designing and synthesizing specific primers and probes used in the detection method of the Docus cedar beetle of the present invention.

[0043] Using MEGA6.0 software, D. pseudotsugae, D. frontalis, D. valens, D. rufipennis, D. armandi, D. black beetle D.terebrans, D.adjunctus, D.approximatus, D.brevicomis, D.jeffreyi, D.micans, D. Murrayanae, D.ponderosae, D.punctatus, D.simplex, D.mexicanus, D.parallelocollis, D.rhizophagus mtCOI gene (mitochondrial DNA) sequences were compared. According to the comparison results, the specific sites of Douglas ch...

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Abstract

The invention provides a method for detecting dendroctonus pseudotsugae. The aim of identifying dendroctonus pseudotsugae can be achieved by combining specific primers (HS-F and HS-R) and a probe (HS-P) which are screened by a test in a Taq-Man real-time fluorescent quantitative PCR (Polymerase Chain Reaction) method. The detection method is suitable for quarantine identification of dendroctonus in different states, the defects of a conventional morphological identification method are overcome, and compared with a DNA bar code identification technology applied in the prior art, the primers and the MGB probe are high in specificity and sensitivity, simple in experimental operation and short in consumed time; the identification result is visual and can be obtained by observing an amplification curve; and the research not only can meet the original rapid customs clearance requirement of entry and exit, improve the detection rate, but also can effectively prevent invasion and diffusion of dangerous dendroctonus, and has important significance on protection of agriculture and forestry production and ecological safety in China.

Description

technical field [0001] The invention belongs to the technical field of molecular identification of biological species, and relates to a real-time fluorescent PCR detection method for the beetle, in particular to the detection method and the application thereof; in addition, the present invention also relates to primers and probes for detecting the beetle. Needle. Background technique [0002] The genus Dendroctonus belongs to Coleoptera, Curculionidae, Scolytinae, and is an important dry-boring pest in forest ecosystems. It mainly causes damage to trees such as Pinus, Picea, and Douglas. This genus (non-Chinese species) has been included in the list of imported plant quarantine pests in my country. There are a total of 20 known species of the genus Glycerus in the world, of which 18 species are distributed in Central and North America, and 3 species are currently distributed in Europe and Asia. Dendroctonuspseudotsugae is a non-Chinese species of the genus Dendroctonuspseud...

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/6888C12Q1/686C12Q2561/113C12Q2563/107C12Q2545/114C12Q2561/101
Inventor 蔡波孟瑞刘嘉欢徐卫敖苏刘福秀韩玉春潘英文
Owner 海口海关热带植物隔离检疫中心
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