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Molecular markers for the identification of C. changling and their applications

A technology of molecular markers and nematodes, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., can solve the problems of poor identification effect and high technical requirements, and achieve simple detection methods and high identification accuracy , the effect of fast detection speed

Active Publication Date: 2020-04-10
INST OF PLANT PROTECTION HEBEI ACAD OF AGRI & FORESTRY SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the problems of high technical requirements for operators in the morphological identification of C. changlingensis, and the poor effect of PCR detection and identification by using universal primers, the purpose of the present invention is to provide a molecular marker for identifying C. changlingensis

Method used

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  • Molecular markers for the identification of C. changling and their applications
  • Molecular markers for the identification of C. changling and their applications
  • Molecular markers for the identification of C. changling and their applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Embodiment 1, the PCR detection test of the design and primer-specificity of the present invention's chrysanthemum-specific primer

[0046] Proceed as follows:

[0047] (1) Test materials and sources

[0048] 1. Changling hair pad nematode: HEB1, HEB2, HEB3, SY1, CC1, QD1, ZB1 and XT1, a total of 8 species.

[0049] 2. Other nematodes: small rod nematode C1, stem nematode D1, root-knot nematode M1, cyst nematode H1 and brevis nematode P1.

[0050] The above nematodes were all preserved in the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences.

[0051] (2) Test method

[0052] (1) DNA extraction from a single nematode. Wash the nematodes in sterilized deionized water, pick a single nematode and put it into 10 μL lysis buffer (1×PCR Buffer (Mg 2+ ), 1 μg proteinase K), 65°C water bath for 1min, liquid nitrogen treatment for 2min, repeated freezing and thawing several times, incubate at 56°C for 20min, and heat at 95°C for 10min to obta...

Embodiment 2

[0062] Embodiment 2, double-PCR method of the present invention detects the specificity test of Changling hairpin nematode

[0063] (1) Test material: (1) with embodiment 1.

[0064] (2) Test method:

[0065] (1). DNA extraction from a single nematode. With (two) (1) of embodiment 1.

[0066] (2). Double PCR amplification. Using nematode DNA as a template and TC-F1 / TC-R1, D2A / D3B as primers, double PCR amplification was performed to obtain the amplified product. The PCR reaction system is 25 μL: 10×Ex Taq Buffer (Mg 2+ ) 2.5 μL, 5U / μL Ex Taq polymerase 0.2 μL, template DNA 0.5 μL, 2.5 mmol / L dNTPs Mixture 2 μL, primer TC-F1 / TC-R1 0.5 μL each, D2A / D3B (10 μmol L -1 ) each 1 μL, and finally make up ddH2O to 25 μL, and set a negative control without template. PCR reaction program: 95°C for 4min; 94°C for 30s, 62°C for 45s, 72°C for 45s, 35 cycles; 72°C for 10min; 16°C for storage. The primers D2A and D3B described therein are universal primers for the 28S region as interna...

Embodiment 3

[0072] Embodiment 3, double PCR method detection sensitivity test of the present invention

[0073] (1) Test materials

[0074] A single nematode DNA was diluted in a concentration gradient according to 1 / 4×, 1 / 8×, 1 / 16×, 1 / 32×, 1 / 64×, 1 / 128×, 1 / 256×, 1 / 512×.

[0075] (2) Test method

[0076] (1). The DNA extraction method of HEB1 single nematode is the same as that in Example 1.

[0077] (2). The double PCR amplification method is the same as in Example 2.

[0078] (3). Electrophoretic detection is the same as in Example 1.

[0079] Results (see image 3 ) single nematode DNA is 1 / 4×(concentration 1ng / μL) to 1 / 32×(concentration 0.125ng / μL) (see image 3 Swimming lanes 1-4), clear and stable bands can be amplified, that is, detection can be carried out at a very low template concentration of 0.125ng / μL, indicating that the double PCR detection method of the present invention has high sensitivity.

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Abstract

The invention discloses a molecular marker for identifying trichotylenchus changlingensis, belongs to the field of plant nematode identification, and the molecular marker is a DNA segment of 520bp. The invention also discloses a PCR (polymerase chain reaction) primer of the molecular marker which is formed by the nucleotide sequences shown as the SEQ ID No. 2 and SEQ ID No. 3. The invention further discloses a primer and a molecular marker of double PCR for identifying the trichotylenchus changlingensis, and a corresponding double PCR identification method. The primer designed by aiming at the trichotylenchus changlingensis, which is provided by the invention, is high in specificity; the molecular marker obtained by amplifying the primer is high in accuracy and reliability when being used for indentifying the trichotylenchus changlingensis; furthermore, the method provided by the invention has the advantages that the detection sensitivity is high, the method is simple, the detection speed is high and the efficiency is high; in addition, the requirement to operators is low.

Description

technical field [0001] The invention belongs to the field of identification of plant nematodes, and in particular relates to a molecular marker for identifying the nematode Changling; it also relates to a specific primer for identifying the nematode, and using the molecular marker or specific primer to identify the nematode of Changling nematode method. Background technique [0002] Trichotylenchus changlingensis is a migratory plant ectoparasitic nematode belonging to the genus Trichotylenchus. The nematode was first discovered in potato rhizosphere soil in 2011 (Xu CL et al., Nematology, 2011, 13(1):45-49). The nematode can cause symptoms such as yellowing of maize leaves, dwarfing of plants, cracking of stem bases, and shortening of stem nodes (Guo Ning et al. Acta Plant Protection. 2015, 42(6):884-8910), which is very harmful to maize. [0003] There are few domestic and foreign studies on C. changlingensis. The traditional identification method is mainly based on morp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6888C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6888C12Q2600/16C12Q2537/143
Inventor 郭宁石洁马红霞刘树森张海剑赵宝广
Owner INST OF PLANT PROTECTION HEBEI ACAD OF AGRI & FORESTRY SCI
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