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RPA primer, probe and kit for rapidly detecting Heterodera filipjevi, and application of RPA primer and probe

A technology of Philips spore and cyst nematode, applied in the fields of biochemical equipment and methods, DNA/RNA fragment, recombinant DNA technology, etc., to achieve the effect of fast reaction speed, low equipment cost and high sensitivity

Active Publication Date: 2022-05-17
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, no studies have been reported using RPA technology to detect Phillips cyst nematode

Method used

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  • RPA primer, probe and kit for rapidly detecting Heterodera filipjevi, and application of RPA primer and probe
  • RPA primer, probe and kit for rapidly detecting Heterodera filipjevi, and application of RPA primer and probe
  • RPA primer, probe and kit for rapidly detecting Heterodera filipjevi, and application of RPA primer and probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] The extraction of embodiment 1 Philip's cyst nematode DNA

[0043] Pick a single second-instar larva or cyst into a 0.2 mL PCR tube, add 20 μL DNA isolation buffer, including 10 μL double-distilled water (ddH 2 O), 7 μL PCR buffer [100 mM Tris-HCl (pH8.9), 500 mM KCl and 15 mM MgCl 2 ] (Takara-Bio, Shiga, Japan) and 3 μL Protein K [600ng / mL] (Solarbio, Beijing, China), DNA isolation methods such as Peng et al. (Peng H, Qi X, Peng D, et al. Sensitive and Direct Detection of Heterodera filipjevi in ​​Soil and Wheat Roots by Species-Specific SCAR-PCRAssays.[J].Plant Disease, 2013,2(23):1-28). Pure genomic DNA was isolated from 5000 J2 using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following the commercial instructions. According to the product instructions, use Soil DNA Isolation Kit (No.12988-10, Qiagen) was used to extract soil DNA (sDNA) from artificially inoculated soil and natural soil in the field. DNA was quantified using a Nano Drop ND-2000 spectrop...

Embodiment 2

[0044] Example 2 Establishment of RPA technology detection method for Philip's cyst nematode H.filipjevi

[0045] RPA primers and probes were designed according to the instructions of the DNA Constant Temperature Rapid Amplification Kit.

[0046] Primer design: A set of primers HfRPA-F3 and HfRPA-R3 were designed according to the SCAR-specific sequence (KC529338) of H. filipjevi, and the 5' end of the downstream primer was labeled with a modified gene (commonly used biotin). Fluorescent probe design: label the 5' end with a fluorescent group; in the middle of the upstream and downstream primers, design a 50bp complementary sequence to the target fragment. A tetrahydrofuran (THF) is marked in the middle of the 5' end and 3' end as the recognition site of nfo; the 3' end is marked with a modified gene (amine group, phosphate group or Spacer-C3 phosphoramidite).

[0047] Table 2 RPA primers and probes

[0048]

[0049] Using the specific primers and probes in Table 2, the H....

Embodiment 3

[0052] Example 3 Establishment and optimization of RPA-LFD system of Phillips cyst nematode.

[0053] On the basis of Example 2, 8 different reaction temperatures (15, 20, 25, 30, 35, 40, 45 and 50°C) and 7 reaction times (1, 5, 10, 15, 20, 25 and 30min). In order to further determine the optimal reaction temperature, a single second-instar larva of Phillips cyst nematode was used as a template, and the reaction was carried out at 8 different temperatures of 35, 36, 37, 38, 39, 40, 41 and 42°C. RPA amplification.

[0054] Such as figure 1 As shown, the results show that the RPA detection of Philip's cyst nematode can observe electrophoretic bands in the temperature range of 25 ° C to 45 ° C ( figure 1 A). Between 35°C and 42°C, the brightness of the bands did not increase significantly ( figure 1 B). In addition, seven RPA reactions were carried out at 40°C for different times, and the results showed that when the reaction time was 5 to 30 minutes, bands could be seen ( ...

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Abstract

The invention discloses RPA primers and probes for rapidly detecting heterodera filipjevi, a kit and application, and belongs to the field of detection of plant parasitic nematodes. The RPA primer is as shown in SEQ ID NO: 1-2, and the probe is as shown in SEQ ID NO: 3. The RPA detection method for Heterodera filipjevi is established by using the RPA primer and the probe, the method is high in sensitivity and very strong in specificity, the detection threshold is 10-3 second-stage larva DNA, 10-4 single cyst DNA and 0.01 ng genome DNA, and the sensitivity is 100 times that of common PCR detection; no cross reaction exists between the nematodes and common closely related plant parasitic nematodes. The whole RPA detection process can be completed within one hour. The detection method is strong in specificity, high in sensitivity, visual in result, rapid and accurate, and has very high application value in the aspects of early diagnosis, field detection and early warning and the like of the Heterosporidium filipjevi disease.

Description

technical field [0001] The invention relates to the field of detection of plant parasitic nematodes, in particular to an RPA primer, probe, kit and application for rapid detection of Phillips cyst nematodes. Background technique [0002] Cereal cyst nematodes are a large group of cyst nematodes that seriously affect cereal crops such as wheat, barley, and oats. They are distributed and harmed in major wheat producing areas in the world, causing major yield losses of wheat Among them, cereal cyst nematodes (Heterodera avenae), Phillip cyst nematodes (H.filipjevi) and wheat cyst nematodes (H.latipons) are the most harmful. [0003] The morphological differences of cyst nematodes are very small, mainly due to the structure of the cyst vulva cone, and there are also subtle differences in the color of cysts, vesicles, and the shape of the double-membrane pores, but these differences require extensive professional knowledge to be able to distinguish them. It is difficult to apply...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6888C12Q1/6844C12N15/11
CPCC12Q1/6888C12Q1/6844C12Q2531/119C12Q2563/107C12Q2565/125C12Q2521/507C12Q2522/101C12Q2537/1376C12Q2565/625Y02A50/30C12Q2600/158
Inventor 彭德良彭焕邵蝴蝶黄文坤孔令安
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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