RPA primers for identifying Ditylenchus destructor and application thereof

A technique for stem nematode and potato, which is applied in the field of RPA primers for identifying D. destructor potato, can solve the problems that D. destructor potato has not been found, and achieve the effects of fast detection speed, low reaction temperature and easy operation

Active Publication Date: 2020-03-06
INST OF PLANT PROTECTION HEBEI ACAD OF AGRI & FORESTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • RPA primers for identifying Ditylenchus destructor and application thereof
  • RPA primers for identifying Ditylenchus destructor and application thereof
  • RPA primers for identifying Ditylenchus destructor and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Embodiment 1, the design and screening of D. destructor RPA primers and probes

[0038] (1) Design of RPA primers and fluorescent probes

[0039] Based on the conserved region of the rDNA-ITS sequence of Ditylenchus destructor (Ditylenchus destructor), five upstream primers, five downstream primers, and one probe were designed according to the RPA primer design principle. The specific sequence information is shown in Table 1.

[0040] Table 1. RPA primer and probe sequence design list

[0041]

[0042] Among them: FAM-dT is a thymine nucleotide carrying a fluorescein group, THF is tetrahydrofuran, BHQ1-dT is a thymine nucleotide carrying a fluorescence quenching group BHQ1, and C3-Spacer is a nucleotide at the 3′ end A spacer arm is introduced to prevent chain extension.

[0043] (2) Extraction of D. destructor DNA

[0044] Single nematode DNA extraction: extract according to the method described in the invention patent CN109750034A.

[0045] A large amount of ne...

Embodiment 2

[0054] Embodiment 2 RPA primer of the present invention is to the specificity detection test of potato rot nematode

[0055] (1) Nematodes to be tested: Ditylenchus destructor (including three groups A, B and C), root-knot nematode (Meloidogyne incognita), cereal cyst nematode (Heterodera avenae), and short-body nematodes (Pratylenchus neglectus), Steinernema feltiae, and Heterorhabditisbacteriophora, all of which are preserved in the Institute of Plant Protection, Hebei Academy of Agriculture and Forestry Sciences.

[0056] (2) DNA preparation: the single nematode genomic DNA of the nematode described in step (1) was respectively extracted by the method described in step (2) of Example 1.

[0057] (3) RPA reaction system: according to According to the instructions of the exo kit, add the following components in sequence to the reaction tube containing RPA lyophilized powder: 29.5 μL of rehydration buffer, 2.1 μL of upstream primer DtITS-F4 (10 μM), and 2.1 μL of downstream ...

Embodiment 3

[0060] The sensitivity test of embodiment 3 RPA primer set of the present invention

[0061] Proceed as follows:

[0062] (1) DNA preparation: extract a single nematode genomic DNA of D. destructor potato by the method described in step (2) of Example 1, the total system is 10 μL, that is, the initial concentration is 0.1 head / μL, and it is carried out with a 5-fold gradient dilution method Dilution, respectively diluted to the original concentration: 1 / 5, 1 / 5 2 , 1 / 5 3 , 1 / 5 4 , 1 / 5 5 , 1 / 5 6 , 1 / 5 7 .

[0063] (2) RPA reaction system: refer to step (4) of Example 2, and 2 μL of genomic DNA of each gradient concentration was added to the template accordingly.

[0064] (3) RPA reaction conditions: see step (4) of Example 2.

[0065] Results (see Figure 5 ) at 0.1×1 / 5 head / μL and 0.1×1 / 5 2 The amplification curve appears at the concentration of head / μL, indicating that the detection limit of this detection system is 0.1×1 / 5 2 head / μL×2μL=1 / 5 3 The DNA of 1 / 125 head...

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Abstract

The invention discloses an RPA primer group for identifying Ditylenchus destructor. The RPA primer group comprises primers and a probe, wherein the primers consist of nucleotide sequences shown as SEQID No. 1 and SEQ ID No. 2, and the probe consists of nucleotide sequences shown as SEQ ID No. 3. The invention also discloses a detection kit containing the primer group and a constant-temperature fluorescence amplification detection method for identifying the Ditylenchus destructor by utilizing the primer group. The RPA primer group and the detection method thereof provided by the invention havethe advantages of strong specificity, high sensitivity, high detection speed, low reaction temperature, simple operation and low cost when being used for detecting the Ditylenchus destructor, and canmeet the requirements of port quarantine and field real-time detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an RPA primer for identifying D. destructor potato; it also relates to the application of the RPA primer. Background technique [0002] Ditylenchus destructor (Ditylenchus destructor; alias: Ditylenchus destructor, Ditylenchus destructor, Ditylenchus destructor, Ditylenchus destructor, etc.) is an important migratory plant endoparasitic nematode, which mainly damages the underground parts of plants, especially roots and tubers. and bulbs etc. This nematode is not only very serious damage to potato, but also causes serious damage to sweet potato, even destructive damage, such as the sweet potato diseased field of this nematode in many provinces and cities in my country can generally cause a 10% to 30% reduction in yield, and it can reach as high as 10%. 50% to 60%, or even no harvest, so the damage to sweet potato is more serious; in addition, the host of this nematode als...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2563/107C12Q2525/186C12Q2521/507
Inventor 高波马娟李秀花王容燕陈书龙
Owner INST OF PLANT PROTECTION HEBEI ACAD OF AGRI & FORESTRY SCI
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