Detection kit for pine wood nematode and detection method therefor

A technology of pine wood nematodes and probes, which is applied in the field of quarantine of plant diseases and insect pests, can solve the problems of difficult identification, small number of nematodes, and easy cross-contamination, so as to avoid pollution and damage to the human body, shorten detection time, and reduce cross-contamination Effect

Inactive Publication Date: 2006-12-20
上海市林业病虫防治检疫站 +1
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Problems solved by technology

[0005] At present, the main quarantine method for pine wood nematodes at ports is morphological observation, but morphological observations have the following defects: First, pine wood nematodes intercepted at ports generally exist in the wooden packaging boards or backing boards of goods, and these materials are generally relatively dry , the number of nematodes is small, and most of them are larvae, which are often in a dormant state. Their stylets and esophagus have degenerated. It is difficult to directly identify t

Method used

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  • Detection kit for pine wood nematode and detection method therefor
  • Detection kit for pine wood nematode and detection method therefor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Embodiment 1: Identification of pine xylophilus

[0073] Pick a single nematode and place it in a thin-walled tube containing 10ulTE buffer, freeze it in a -70°C refrigerator for 15 minutes, and thaw it at room temperature as a PCR reaction template.

[0074] Set up 18 disposable real-time quantitative capillary PCR tubes:

[0075] Add pine wood nematode template to No. 1-8, add B. xylophilus template to No. 9-14, add 1 ul each of the unnamed templates of 2 species of the genus P. negative control. Add to each tube separately:

[0076] 10x Buffer 1.0ul

[0077] MgCl 2 (25mM) 2.0ul

[0078] dNTP(2.5mM) 1.2ul

[0079] Primer F (10uM) 0.2ul

[0080] Primer R (10uM) 0.2ul

[0081] Probe P (5uM) 0.24ul

[0082] Taq enzyme (5U / ul) 0.1ul

[0083] Heavy steamed ultrapure H 2 0 to 10ul total volume

[0084] The PCR reaction was performed on a Lightcycler fluorescent PCR instrument: denaturation at 95°C for 3 minutes, denaturation at 95°C for 10 secon...

Embodiment 2

[0086] Embodiment 2: sensitivity test

[0087] The DNA of the pine wood nematode (Bxlyg) that will be collected from China Lianyungang is diluted to 1 / 10, 1 / 20, 1 / 40, the concentration gradient of 1 / 80 respectively, detects by above-mentioned method, the result is as follows figure 2 .

[0088] This method can detect the presence of 1 / 80 nematode DNA, while the conventional method requires the entire nematode DNA as a template to obtain the amplified band (the conventional method will not be described in detail here).

[0089] The results show that the method of the invention has good specificity and high sensitivity.

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Abstract

The invention discloses a case of rapid, sensitive, precise pine beam nematology detecting box and method, which consists of TaqMan-MGB probe and mating primer. The method adopts super-low temperature (-70 deg.c) freezing method to extract nematology DNA rapidly as form to proceed PCR augmentation, which judges the detecting result according to fluorescent curve. The invention is convenient to operate within 1.5 h to finish the whole detecting course, which is fit for animal quarantine, plant protection, packing company and forestry department.

Description

technical field [0001] The invention relates to the field of plant quarantine, in particular to a quarantine method for plant diseases and insect pests. Specifically, a method and kit for rapid detection of pine wood nematode using TaqMan-MGB probe real-time fluorescent PCR technology and its kit are provided, which are suitable for applications in plant quarantine, wood packaging production, plant protection and other departments. Background technique [0002] Pine wood nematode is the most dangerous forest disease in the world, and host plants can die within 40 days after being infected with pine xylophilus. Therefore, it is called the "cancer" of pine trees. It only takes 3-5 years from the onset to the destruction of the entire pine forest. The causative nematode of this disease is pine wood nematode (Bursaphelenchus xylophilus), which belongs to the order of the order, the superfamily, and the genus of the genus. At present, there is no good prevention and treatment ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/25
Inventor 王焱林茂松季镭马凤林
Owner 上海市林业病虫防治检疫站
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