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Method for assisting in identifying root-knot nematodes and special primer pair thereof

A technology of root knot nematode and southern root knot nematode, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragment, etc., can solve problems such as unreachable, low SCAR sensitivity, and long time consumption

Inactive Publication Date: 2012-08-08
ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, scholars at home and abroad often use mtDNA-RFLP, rDNA-ITS and SCAR to identify the species of root-knot nematodes. The sensitivity is not strong) and cannot achieve the purpose of rapid and accurate identification

Method used

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  • Method for assisting in identifying root-knot nematodes and special primer pair thereof
  • Method for assisting in identifying root-knot nematodes and special primer pair thereof
  • Method for assisting in identifying root-knot nematodes and special primer pair thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1, the preparation of primer

[0035] Artificially synthesized the following four primers:

[0036] #C2F3: 5'-GGTCAA TGTCAGAAA TTTGTGG-3'; Sequence 1;

[0037] #1108: 5'-TACCTTTGACCAA TCACGCT-3'; Sequence 2;

[0038] MI-F: 5'-GGGCAAGTAAGGATGCTCTGAC-3'; Sequence 3;

[0039] MI-R (5'-CTTTCATAGCCACGTCGCGATC-3'; SEQ ID NO: 4.

[0040] The primer pair consisting of #C2F3 and #1108 was used as primer pair A, and the primer pair consisting of MI-F and MI-R was used as primer pair B. Primer pair A targets the region between CO II and LrRNA in mitochondrial DNA (mtDNA). Primer pair B is directed against the esophageal adenin gene.

Embodiment 2

[0041] Embodiment 2, application primer is assisted in distinguishing root-knot nematode (adult)

[0042] 1. Using primers to help identify root-knot nematodes

[0043] 1. Extract the genomic DNA of a single root-knot nematode (adult).

[0044] 2. Using genomic DNA as a template to carry out PCR reaction to obtain PCR amplification products.

[0045] PCR reaction system (25 μL): 17.1 μl ddH20; 2.5 μl 10× buffer; 2 μl dNTP (2.5 mM); 1 μl primer #C2F3 (20 μM); 1 μl primer #1108 (20 μM); 0.4 μl Taq enzyme (5U / μl); 1 μl template.

[0046] PCR reaction program: pre-denaturation at 94°C for 4 minutes; 40 cycles of denaturation at 94°C for 1 minute, annealing at 48°C for 1 minute, and extension at 72°C for 2 minutes; finally, incubation at 72°C for 5 minutes.

[0047] 3. The PCR amplification product was subjected to 1% (containing 0.5 μg / mL EB) agarose gel electrophoresis, the electrophoresis buffer was 0.5×TBE, the voltage was 80 V, and the electrophoresis was about 30 min. Pho...

Embodiment 3

[0056] Embodiment 3, application primer pair assists in identifying root-knot nematode larvae (2 instar larvae)

[0057] 1. Using primers to help identify root-knot nematodes

[0058] 1. Extract the genomic DNA of a single root-knot nematode (2nd instar larva).

[0059] 2. Using genomic DNA as a template to carry out PCR reaction to obtain PCR amplification products.

[0060] PCR reaction system (25 μL): 17.1 μl ddH20; 2.5 μl 10× buffer; 2 μl dNTP (2.5 mM); 1 μl primer #C2F3 (20 μM); 1 μl primer #1108 (20 μM); 0.4 μl Taq enzyme (5U / μl); 1 μl template.

[0061] PCR reaction program: pre-denaturation at 94°C for 4 minutes; 40 cycles of denaturation at 94°C for 1 minute, annealing at 48°C for 1 minute, and extension at 72°C for 2 minutes; finally, incubation at 72°C for 5 minutes.

[0062] 3. The PCR amplification product was subjected to 1% (containing 0.5 μg / mL EB) agarose gel electrophoresis, the electrophoresis buffer was 0.5×TBE, the voltage was 80 V, and the electrophore...

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Abstract

The invention discloses a method for assisting in identifying root-knot nematodes and a special primer pair thereof. The invention provides a special primer consisting of a primer pair A and a primer pair B, wherein the primer pair A consists of DNA (Deoxyribose Nucleic Acid) shown as a sequence 1 in a sequence table and DNA shown as a sequence 2 in the sequence table; the primer pair B consists of DNA shown as a sequence 3 in the sequence table and DNA shown as a sequence 4 in the sequence table; and the special primer can be used for assisting in identifying the root-knot nematodes. The method provided by the invention can be used for correctly distinguishing four common root-knot nematodes, has high sensitivity, and can be used for identifying the types of root-knot nematodes by using a single egg capsule or a single second stage juvenile. The root-knot nematode can be directly separated from diseased tissues without purification culturing, the purification culturing time of the nematodes can be greatly shortened, and the detection efficiency can be improved.

Description

technical field [0001] The invention relates to a method for assisting identification of root-knot nematodes and a pair of special primers. Background technique [0002] Plant parasitic nematode is a common plant disease worldwide, harming more than 3000 kinds of plants. Worldwide losses due to nematodes are more than $100 billion per year. Plant root-knot nematode diseases occur widely in my country, especially in southern regions, such as banana root-knot nematode, watermelon root-knot nematode, and pepper root-knot nematode. Since there are significant differences in pathogenicity among root-knot nematode species, successful applications such as crop resistance and plant quarantine rely on rapid and accurate identification of root-knot nematode species. basis of prevention. [0003] At present, scholars at home and abroad often use mtDNA-RFLP, rDNA-ITS and SCAR to identify the species of root-knot nematodes. The sensitivity is not strong) and cannot achieve the purpos...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12Q1/68
Inventor 黄俊生景晓辉汪军吴伦英吴琳薛玉潇朱利林杨腊英
Owner ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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