RPA (recombinase polymerase amplification) primer, probe and kit for detecting heterodera avenae and application

A cereal cyst nematode technology and a kit are applied in the fields of biochemical equipment and methods, DNA/RNA fragments, recombinant DNA technology, etc., which can solve the problems such as no report on the cereal cyst nematode, and achieve intuitive sensitivity and specificity. Strong and sensitive effect

Active Publication Date: 2022-04-29
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, although researchers have applied this method to the detection of plant parasitic ne

Method used

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  • RPA (recombinase polymerase amplification) primer, probe and kit for detecting heterodera avenae and application
  • RPA (recombinase polymerase amplification) primer, probe and kit for detecting heterodera avenae and application
  • RPA (recombinase polymerase amplification) primer, probe and kit for detecting heterodera avenae and application

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Experimental program
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Embodiment 1

[0038] The extraction of embodiment 1 cereal cyst nematode DNA

[0039] 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 ofHeterodera filipjevi in ​​Soil and Wheat Roots by Species-Specific SCAR-PCRAssays.[J].Plant Disease, 2013,2(23):1-28), using DNeasy Blood&Tissue Kit (Qiagen, Hilden, Germany), according to manufacture Pure genomic DNA was isolated from 5000 second instar larvae according to the description of the merchant. 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...

Embodiment 2

[0040] The establishment of embodiment 2RPA technique detection gramineous cyst nematode method

[0041] RPA primers and probes were designed according to the instructions of the DNA Constant Temperature Rapid Amplification Kit (see Table 2).

[0042] Primer design: Design a sequence with a length of 100bp-300bp according to the specific SCAR sequence (JQ405270.1) of the cereal cyst nematode genome, and mark a modified gene (commonly used biotin) at the 5' end of the downstream primer. Fluorescent probe design: Between the upstream and downstream primers, design a 46-52bp complementary sequence to the target fragment. The 5' end is labeled with a fluorescent group, and the middle position of the 5' and 3' ends is marked with a dSpacer (THF) as the recognition site of nfo; the 3' end is marked with a modified gene (amine group, phosphate group or C3- Spacer phosphoramidite).

[0043] Table 2 Primers, probes and amplified product sequences

[0044]

[0045] Using the speci...

Embodiment 3

[0047] The establishment and optimization of embodiment 3RPA-LFD system

[0048] 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 30 min). In order to further determine the optimal reaction temperature, RPA was carried out at 8 different temperatures of 35, 36, 37, 38, 39, 40, 41 and 42°C, using a single second-instar larva of the cereal cyst nematode H. avenae as a template Amplify.

[0049] The result is as figure 1 As shown, bands can be observed in a wide temperature range from 15°C to 50°C during electrophoresis detection ( figure 1 A). Between 35°C and 40°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 ( figure 1 C), the optimum reaction time is 10 to 30 minutes.

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Abstract

The invention discloses an RPA primer, a probe and a kit for detecting heterodera avenae and application, and belongs to the field of detection of plant parasitic nematodes. The RPA primer and the probe comprise a pair of specific primers HaRPA-F3 and HaRPA-R3 and a probe Ha-probe, the nucleotide sequences of the HaRPA-F3 and the HaRPA-R3 are as shown in SEQ ID NO: 1 to SEQ ID NO: 2, and the nucleotide sequence of the Ha-probe is as shown in SEQ ID NO: 3. The rapid visual detection method for the heterodera avenae is established by using the RPA primer and the probe, the sensitivity is high, the specificity is strong, and the whole detection process can be completed within 1 hour. Therefore, the RPA detection method disclosed by the invention is a simple, rapid, specific, high-sensitivity and intuitive method, and can be suitable for rapidly detecting the heterodera avenae in the field.

Description

technical field [0001] The invention relates to the detection field of plant parasitic nematodes, in particular to a primer, a probe, a kit and an application for detecting the RPA of the cereal cyst nematode H. avenae. 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 , the most serious of which are cereal cyst nematodes (Heterodera avenae), Philip cyst nematodes (Heterodera filipjevi) and wheat cyst nematodes (Heterodera latipons). [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 the...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/11C12Q1/6844
CPCC12Q1/6888C12Q1/6844C12Q2521/507C12Q2521/119C12Q2522/101C12Q2563/107C12Q2565/625C12Q2565/125Y02A50/30
Inventor 彭焕彭德良邵蝴蝶黄文坤孔令安
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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