Test strip RPA primer and detection kit for detecting Ditylenchus destructor

A detection kit, D. nematode technology, applied in the directions of DNA/RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problems of difficult to meet the requirements of on-site real-time detection and high requirements for personnel quality, and achieve convenient on-site detection. , The effect of simple equipment requirements and low reaction temperature

Pending Publication Date: 2020-09-18
INST OF PLANT PROTECTION HEBEI ACAD OF AGRI & FORESTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] Aiming at the existing PCR, LAMP and real-time fluorescent RPA technologies have high requirements for equipment and personnel quality, and are difficult to meet the requirements of on-site real-time detection, the purpose of the present invention is to detect D.

Method used

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  • Test strip RPA primer and detection kit for detecting Ditylenchus destructor
  • Test strip RPA primer and detection kit for detecting Ditylenchus destructor
  • Test strip RPA primer and detection kit for detecting Ditylenchus destructor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The design and screening test of test strip RPA primer and probe of embodiment 1 D. destructor potato

[0042] (1) According to the design principles of RPA primers and probes on test strips, based on the conserved region of the rDNA-ITS sequence of Ditylenchus destructor (Ditylenchus destructor), the following 5 upstream primers, 6 downstream primers and a probe were designed (both provided by Shanghai Sangon Synthetic) (see Table 1):

[0043] Table 1. LFD-RPA primer and probe sequence design list

[0044]

[0045] Among them, Biotin is to introduce a biotin group at the 5' end of the downstream primer; FAM is to introduce a fluorescein group at the 5' end of the probe; THF is tetrahydrofuran; C3-Spacer is used to introduce a spacer at the 3' end to prevent the chain extension.

[0046] (2) Extraction of D. destructor DNA:

[0047] (a) Extraction of single nematode DNA: extract according to the method of the invention patent CN109750034A.

[0048] (b) Extraction...

Embodiment 2

[0056] The specificity test of embodiment 2 test strip RPA candidate primer combination to D. destructor

[0057](1) The present inventor has disclosed a set of primers and probes for real-time fluorescent RPA for detecting D. destructor in potato in patent CN110863058A. In order to verify whether the primer-probe combination is applicable to the test strip RPA, the present invention re-base-modified according to the requirements of the test strip RPA on the basis of the sequence of the primer-probe combination (see Table 2).

[0058] Primer and probe combination after table 2 base modification

[0059]

[0060] (2) Test method:

[0061] (1) Nematodes to be tested: Ditylenchus destructor, Meloidogyne enterolobii, Meloidogyne hapla, Meloidogyne incognita, Phillips cyst nematode ( Heterodera filipjevi), Heterodera avenae, Pratylenchus zeae, Steinernemafeltiae, Heterorhabditis bacteriophora, Trichotylenchus changlingensis 1. Ditylenchus spp. in soil, all of which are stored...

Embodiment 3

[0070] The sensitivity test of embodiment 3 test strip RPA primer set of the present invention

[0071] Proceed as follows:

[0072] (1) DNA preparation: extract a single nematode genomic DNA of D. destructor in the method described in step (2) of Example 2, 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 .

[0073] (2) RPA reaction system and reaction conditions: refer to steps (3) and (4) of Example 2, respectively, and 2 μL of genomic DNA of each gradient concentration was added to the template.

[0074] Results (see Figure 6 ) at 1 / 5, 1 / 5 2 , 1 / 5 3 , 1 / 5 4 Under the dilution multiples of the T line, there are obvious red bands, indicating that the detection limit of the detection method of the present invention is 0.1 head / μL×1 / 5 4 ×2μL=1 / 5 5 The DNA of 1 / 312...

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Abstract

The invention discloses a test strip RPA primer for detecting Ditylenchus destructor. The primer is composed of nucleotide sequences as shown in SEQ ID No:1 and SEQ ID No:2. The invention further discloses a combination of the primer and a probe, and the probe is composed of a sequence as shown in SEQ ID No:3. In addition, the invention discloses a detection kit comprising the primer and probe combination. The combination of the test strip RPA primer and the probe has the advantages of strong detection specificity for the Ditylenchus destructor, high sensitivity, low requirements on equipmentand personnel quality, simplicity in operation, high detection speed and low cost, and is suitable for port quarantine and basic level on-site detection.

Description

technical field [0001] The invention belongs to the technical field of nematode detection, and in particular relates to a test strip RPA primer for detecting D. destructor potato; a detection kit containing the primer and their application in detecting D. rotten potato. Background technique [0002] Ditylenchus destructor (Ditylenchus destructor; alias: Ditylenchus destructor, Ditylenchus sweetpotato or Ditylenchus potato, etc.) is a migratory plant parasitic nematode, which mainly damages the underground parts of plants, especially roots, tubers or bulbs. The nematode is not only serious damage to potato, but also very serious damage to sweet potato. In some areas of our country, the yield reduction of sweet potatoes caused by the nematode is generally 10% to 30%, and in severe cases, it can reach 50% to 60%, or even no production. In addition, the nematode has a wide range of damage, and its hosts include more than 90 kinds of plants such as carrots, ginseng, wheat, corn ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6844C12Q1/6888C12Q2531/119C12Q2521/507C12Q2537/1376C12Q2565/625C12Q2563/107
Inventor 高波马娟李秀花李焦生王容燕陈书龙
Owner INST OF PLANT PROTECTION HEBEI ACAD OF AGRI & FORESTRY SCI
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